Pyrrolobenzodiazepines

ABSTRACT

Compounds of the formula I: 
                         
or solvate thereof, wherein:
     R 2  is an optionally substituted C 5-20  aryl group;   R 6  and R 9  are independently selected from H, R, OH, OR, SH, SR, NH 2 , NHR, NRR′, nitro, Me 3 Sn and halo;   where R and R′ are independently selected from optionally substituted C 1-12  alkyl, C 3-20  heterocyclyl and C 5-20  aryl groups;   R 7  is selected from H, R, OH, OR, SH, SR, NH 2 , NHR, NHRR′, nitro, Me 3 Sn and halo;   R″ is a C 3-12  alkylene group, which chain may be interrupted by one or more heteroatoms and/or aromatic rings;   X is selected from O, S, or NH;   z is 2 or 3;   M is a monovalent pharmaceutically acceptable cation;   R 2′ , R 6′ , R 7′ , R 9′ , X′ and M′ are selected from the same groups as R 2 , R 6 , R 7 , R 9 , X and M respectively, or M and M′ may together represent a divalent pharmaceutically acceptable cation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of U.S. application Ser. No.11/911,890, filed Oct. 18, 2007, now U.S. Pat. No. 7,612,062, which is anational stage filing under 35 U.S.C. 371 of International ApplicationNo. PCT/GB2006/001456, filed Apr. 21, 2006, which claims foreignpriority benefits to United Kingdom Application No. 0522746.7, filedNov. 7, 2005 and United Kingdom Application No. 0508084.1, filed Apr.21, 2005. These applications are incorporated herein by reference intheir entireties.

The present invention relates to pyrrolobenzodiazepines (PBDs), and inparticular pyrrolobenzodiazepine dimers bearing C2 aryl substitutions.

BACKGROUND TO THE INVENTION

Some pyrrolobenzodiazepines (PBDs) have the ability to recognize andbond to specific sequences of DNA; the preferred sequence is PuGPu. Thefirst PBD antitumour antibiotic, anthramycin, was discovered in 1965(Leimgruber, et al., J. Am. Chem. Soc., 87, 5793-5795 (1965);Leimgruber, et al., J. Am. Chem. Soc., 87, 5791-5793 (1965)). Sincethen, a number of naturally occurring PBDs have been reported, and over10 synthetic routes have been developed to a variety of analogues(Thurston, et al., Chem. Rev. 1994, 433-465 (1994)). Family membersinclude abbeymycin (Hochlowski, et al., J. Antibiotics, 40, 145-148(1987)), chicamycin (Konishi, et al., J. Antibiotics, 37, 200-206(1984)), DC-81 (Japanese Patent 58-180 487; Thurston, et al., Chem.Brit., 26, 767-772 (1990); Bose, et al., Tetrahedron, 48, 751-758(1992)), mazethramycin (Kuminoto, et al., J. Antibiotics, 33, 665-667(1980)), neothramycins A and B (Takeuchi, et al., J. Antibiotics, 29,93-96 (1976)), porothramycin (Tsunakawa, et al., J. Antibiotics, 41,1366-1373 (1988)), prothracarcin (Shimizu, et al, J. Antibiotics, 29,2492-2503 (1982); Langley and Thurston, J. Org. Chem., 52, 91-97(1987)), sibanomicin (DC-102)(Hara, et al., J. Antibiotics, 41, 702-704(1988); Itoh, et al., J. Antibiotics, 41, 1281-1284 (1988)), sibiromycin(Leber, et al., J. Am. Chem. Soc., 110, 2992-2993 (1988)) and tomamycin(Arima, et al., J. Antibiotics, 25, 437-444 (1972)). PBDs are of thegeneral structure:

They differ in the number, type and position of substituents, in boththeir aromatic A rings and pyrrolo C rings, and in the degree ofsaturation of the C ring. In the B-ring there is either an imine (N═C),a carbinolamine(NH—CH(OH)), or a carbinolamine methyl ether (NH—CH(OMe))at the N10-C11 position which is the electrophilic centre responsiblefor alkylating DNA. All of the known natural products have an(S)-configuration at the chiral C11a position which provides them with aright-handed twist when viewed from the C ring towards the A ring. Thisgives them the appropriate three-dimensional shape for isohelicity withthe minor groove of B-form DNA, leading to a snug fit at the bindingsite (Kohn, In Antibiotics III. Springer-Verlag, New York, pp. 3-11(1975); Hurley and Needham-VanDevanter, Acc. Chem. Res., 19, 230-237(1986)). Their ability to form an adduct in the minor groove, enablesthem to interfere with DNA processing, hence their use as antitumouragents.

The present inventors have previously disclosed, in WO 2004/043963,cytotoxic compounds having an aryl group at the C2 position, forexample:

The present inventors have also previously disclosed, in co-pending PCTapplication PCT/GB2005/000768 (published as WO 2005/085251), dimeric PBDcompounds bearing C2 aryl substituents, such as:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of relative tumour volume (RTV) versus time duringtreatment of mice bearing LOX IMVI (human melanoma) with ZC423.

FIG. 2 is a graph of relative tumour volume (RTV) versus time duringtreatment of mice bearing LOX IMVI (human melanoma) with ZC423, withresults presented over a longer period of time.

FIG. 3 is a graph of relative tumour volume (RTV) versus time duringtreatment of mice bearing OVCAR-5 (human ovarian tumour) with ZC423.

DISCLOSURE OF THE INVENTION

The present inventors have encountered some issues with the solubilityof compounds such as ZC-207, which they have resolved by the use of adifferent form of these compounds.

The present invention comprises a compound with the formula I:

or solvate thereof, wherein:R² is an optionally substituted C₅₋₂₀ aryl group;R⁶ and R⁹ are independently selected from H, R, OH, OR, SH, SR, NH₂,NHR, NRR′, nitro, Me₃Sn and halo;where R and R′ are independently selected from optionally substitutedC₁₋₁₂ alkyl, C₃₋₂₀ heterocyclyl and C₅₋₂₀ aryl groups;R⁷ is selected from H, R, OH, OR, SH, SR, NH₂, NHR, NHRR′, nitro, Me₃Snand halo;R″ is a C₃₋₁₂ alkylene group, which chain may be interrupted by one ormore heteroatoms,e.g. O, S, NH, and/or aromatic rings, e.g. benzene or pyridine;X is selected from O, S, or NH;z is 2 or 3;M is a monovalent pharmaceutically acceptable cation;R^(2′), R^(6′), R^(7′), R^(9′), X′ and M′ are selected from the samegroups as R², R⁶, R⁷, R⁹, X and M respectively, or M and M′ may togetherrepresent a divalent pharmaceutically acceptable cation.

Pyrrolobenzodiazepines having an imine bond are known to convert to thedi-carbinolamine form in water, and isolated pyrrolobenzodiazepinesoften exist as a mixture of the imine, mono-carbinolamine anddi-carbinolamine forms. Furthermore, if the compound is isolated as asolid with a mixture of these three forms, the balance between them maychange over time. Although this does not pose a problem foradministration of the compound, it can provide difficulties inaccurately assessing the amount of active substance in a given amount ofpowder. Compounds of the present invention, at least to some extent,overcome this difficulty whilst remaining activity, and are thus suitedto formulation as pharmaceuticals.

Dimeric pyrrolobenzodiazepines offer advantages over monomericpyrrolobenzodiazepines in that they possess the ability to cross-linkDNA in the minor groove, which can lead to an increase in cytotoxicity.

Further aspects of the invention relate to the compounds use in methodsof therapy (particularly in treating proliferative diseases),pharmaceutical compositions comprising the compounds, and their use inthe manufacture of a medicament for the treatment of a proliferativedisease.

DEFINITIONS Substituents

The phrase “optionally substituted” as used herein, pertains to a parentgroup which may be unsubstituted or which may be substituted.

Unless otherwise specified, the term “substituted” as used herein,pertains to a parent group which bears one or more substitutents. Theterm “substituent” is used herein in the conventional sense and refersto a chemical moiety which is covalently attached to, or if appropriate,fused to, a parent group. A wide variety of substituents are well known,and methods for their formation and introduction into a variety ofparent groups are also well known.

Examples of substituents are described in more detail below.

C₁₋₁₂ alkyl: The term “C₁₋₁₂ alkyl” as used herein, pertains to amonovalent moiety obtained by removing a hydrogen atom from a carbonatom of a hydrocarbon compound having from 1 to 12 carbon atoms, whichmay be aliphatic or alicyclic, and which may be saturated or unsaturated(e.g. partially unsaturated, fully unsaturated). Thus, the term “alkyl”includes the sub-classes alkenyl, alkynyl, cycloalkyl, etc., discussedbelow.

Examples of saturated alkyl groups include, but are not limited to,methyl (C₁), ethyl (C₂), propyl (C₃), butyl (C₄), pentyl (C₅), hexyl(C₆) and heptyl (C₇).

Examples of saturated linear alkyl groups include, but are not limitedto, methyl (C₁), ethyl (C₂), n-propyl (C₃), n-butyl (C₄), n-pentyl(amyl) (C₅), n-hexyl (C₆) and n-heptyl (C₇).

Examples of saturated branched alkyl groups include iso-propyl (C₃),iso-butyl (C₄), sec-butyl (C₄), tert-butyl (C₄), iso-pentyl (C₅), andneo-pentyl (C₅).

C₂₋₁₂ Alkenyl: The term “C₂₋₁₂ alkenyl” as used herein, pertains to analkyl group having one or more carbon-carbon double bonds.

Examples of unsaturated alkenyl groups include, but are not limited to,ethenyl (vinyl, —CH═CH₂), 1-propenyl (—CH═CH—CH₃), 2-propenyl (allyl,—CH—CH═CH₂), isopropenyl (1-methylvinyl, —C(CH₃)═CH₂), butenyl (C₄),pentenyl (C₅), and hexenyl (C₆).

C₂₋₁₂ alkynyl: The term “C₂₋₁₂ alkynyl” as used herein, pertains to analkyl group having one or more carbon-carbon triple bonds.

Examples of unsaturated alkynyl groups include, but are not limited to,ethynyl (ethinyl, —C≡CH) and 2-propynyl (propargyl, —CH₂—C≡CH).

C₃₋₁₂ cycloalkyl: The term “C₃₋₁₂ cycloalkyl” as used herein, pertainsto an alkyl group which is also a cyclyl group; that is, a monovalentmoiety obtained by removing a hydrogen atom from an alicyclic ring atomof a cyclic hydrocarbon (carbocyclic) compound, which moiety has from 3to 7 carbon atoms, including from 3 to 7 ring atoms.

Examples of cycloalkyl groups include, but are not limited to, thosederived from:

-   -   saturated monocyclic hydrocarbon compounds:        cyclopropane (C₃), cyclobutane (C₄), cyclopentane (C₅),        cyclohexane (C₆), cycloheptane (C₇), methylcyclopropane (C₄),        dimethylcyclopropane (C₅), methylcyclobutane (C₅),        dimethylcyclobutane (C₆), methylcyclopentane (C₆),        dimethylcyclopentane (C₇) and methylcyclohexane (C₇);    -   unsaturated monocyclic hydrocarbon compounds:        cyclopropene (C₃), cyclobutene (C₄), cyclopentene (C₅),        cyclohexene (C₆), methylcyclopropene (C₄), dimethylcyclopropene        (C₅), methylcyclobutene (C₅), dimethylcyclobutene (C₆),        methylcyclopentene (C₆), dimethylcyclopentene (C₇) and        methylcyclohexene (C₇); and    -   saturated polycyclic hydrocarbon compounds:        norcarane (C₇), norpinane (C₇), norbornane (C₇).

C₃₋₂₀ heterocyclyl: The term “C₃₋₂₀ heterocyclyl” as used herein,pertains to a monovalent moiety obtained by removing a hydrogen atomfrom a ring atom of a heterocyclic compound, which moiety has from 3 to20 ring atoms, of which from 1 to 10 are ring heteroatoms. Preferably,each ring has from 3 to 7 ring atoms, of which from 1 to 4 are ringheteroatoms.

In this context, the prefixes (e.g. C₃₋₂₀, C₃₋₇, C₅₋₆, etc.) denote thenumber of ring atoms, or range of number of ring atoms, whether carbonatoms or heteroatoms. For example, the term “C₅₋₆heterocyclyl”, as usedherein, pertains to a heterocyclyl group having 5 or 6 ring atoms.

Examples of monocyclic heterocyclyl groups include, but are not limitedto, those derived from:

N₁: aziridine (C₃), azetidine (C₄), pyrrolidine (tetrahydropyrrole)(C₅), pyrroline (e.g., 3-pyrroline, 2,5-dihydropyrrole) (C₅), 2H-pyrroleor 3H-pyrrole (isopyrrole, isoazole) (C₅), piperidine (C₆),dihydropyridine (C₆), tetrahydropyridine (C₆), azepine (C₇);O₁: oxirane (C₃), oxetane (C₄), oxolane (tetrahydrofuran) (C₅), oxole(dihydrofuran) (C₅), oxane (tetrahydropyran) (C₆), dihydropyran (C₆),pyran (C₆), oxepin (C₇);S₁: thiirane (C₃), thietane (C₄), thiolane (tetrahydrothiophene) (C₅),thiane (tetrahydrothiopyran) (C₆), thiepane (C₇);O₂: dioxolane (C₅), dioxane (C₆), and dioxepane (C₇);O₃: trioxane (C₆);N₂: imidazolidine (C₅), pyrazolidine (diazolidine) (C₅), imidazoline(C₅), pyrazoline (dihydropyrazole) (C₅), piperazine (C₆);N₁O₁: tetrahydrooxazole (C₅), dihydrooxazole (C₅), tetrahydroisoxazole(C₅), dihydroisoxazole (C₅), morpholine (C₆), tetrahydrooxazine (C₆),dihydrooxazine (C₆), oxazine (C₆);N₁S₁: thiazoline (C₅), thiazolidine (C₅), thiomorpholine (C₆);N₂O₁: oxadiazine (C₆);O₁S₁: oxathiole (C₅) and oxathiane (thioxane) (C₆); and,N₁O₁S₁: oxathiazine (C₆).

Examples of substituted monocyclic heterocyclyl groups include thosederived from saccharides, in cyclic form, for example, furanoses (C₅),such as arabinofuranose, lyxofuranose, ribofuranose, and xylofuranse,and pyranoses (C₆), such as allopyranose, altropyranose, glucopyranose,mannopyranose, gulopyranose, idopyranose, galactopyranose, andtalopyranose.

C₅₋₂₀ aryl: The term “C₅₋₂₀ aryl”, as used herein, pertains to amonovalent moiety obtained by removing a hydrogen atom from an aromaticring atom of an aromatic compound, which moiety has from 3 to 20 ringatoms. Preferably, each ring has from 5 to 7 ring atoms.

In this context, the prefixes (e.g. C₃₋₂₀, C₅₋₇, C₅₋₆, etc.) denote thenumber of ring atoms, or range of number of ring atoms, whether carbonatoms or heteroatoms. For example, the term “C₅₋₆ aryl” as used herein,pertains to an aryl group having 5 or 6 ring atoms.

The ring atoms may be all carbon atoms, as in “carboaryl groups”.

Examples of carboaryl groups include, but are not limited to, thosederived from benzene (i.e. phenyl) (C₆), naphthalene (CO, azulene (CO,anthracene (C₁₄), phenanthrene (C₁₄), naphthacene (CO, and pyrene (C₁₆).

Examples of aryl groups which comprise fused rings, at least one ofwhich is an aromatic ring, include, but are not limited to, groupsderived from indane (e.g. 2,3-dihydro-1H-indene) (C₉), indene (C₉),isoindene (C₉), tetraline (1,2,3,4-tetrahydronaphthalene (C₁₀),acenaphthene (C₁₂), fluorene (C₁₃), phenalene (C₁₃), acephenanthrene(C₁₅), and aceanthrene (C₁₆).

Alternatively, the ring atoms may include one or more heteroatoms, as in“heteroaryl groups”. Examples of monocyclic heteroaryl groups include,but are not limited to, those derived from:

N₁: pyrrole (azole) (C₅), pyridine (azine) (C₆);

O₁: furan (oxole) (C₅);

S₁: thiophene (thiole) (C₅);

N₁O₁: oxazole (C₅), isoxazole (C₅), isoxazine (C₆);

N₂O₁: oxadiazole (furazan) (C₅);

N₃O₁: oxatriazole (C₅);

N₁S₁: thiazole (C₅), isothiazole (C₅);

N₂: imidazole (1,3-diazole) (C₅), pyrazole (1,2-diazole) (C₅),pyridazine (1,2-diazine) (C₆), pyrimidine (1,3-diazine) (C₆) (e.g.,cytosine, thymine, uracil), pyrazine (1,4-diazine) (C₆);

N₃: triazole (C₅), triazine (C₆); and,

N₄: tetrazole (C₅).

Examples of heteroaryl which comprise fused rings, include, but are notlimited to:

-   -   C₉ (with 2 fused rings) derived from benzofuran (O₁),        isobenzofuran (O₁), indole (N₁), isoindole (N₁), indolizine        (N₁), indoline (N₁), isoindoline (N₁), purine (N₄) (e.g.,        adenine, guanine), benzimidazole (N₂), indazole (N₂),        benzoxazole (N₁O₁), benzisoxazole (N₁O₁), benzodioxole (O₂),        benzofurazan (N₂O₁), benzotriazole (N₃), benzothiofuran (S₁),        benzothiazole (N₁S₁), benzothiadiazole (N₂S);    -   C₁₀ (with 2 fused rings) derived from chromene (O₁), isochromene        (O₁), chroman (O₁), isochroman (O₁), benzodioxan (O₂), quinoline        (N₁), isoquinoline (N₁), quinolizine (N₁), benzoxazine (N₁O₁),        benzodiazine (N₂), pyridopyridine (N₂), quinoxaline (N₂),        quinazoline (N₂), cinnoline (N₂), phthalazine (N₂),        naphthyridine (N₂), pteridine (N₄);    -   C₁₁ (with 2 fused rings) derived from benzodiazepine (N₂);    -   C₁₃ (with 3 fused rings) derived from carbazole (N₁),        dibenzofuran (O₁), dibenzothiophene (S₁), carboline (N₂),        perimidine (N₂), pyridoindole (N₂); and,    -   C₁₄ (with 3 fused rings) derived from acridine (N₁), xanthene        (O₁), thioxanthene (S₁), oxanthrene (O₂), phenoxathiin (O₁S₁),        phenazine (N₂), phenoxazine (N₁O₁), phenothiazine (N₁S₁),        thianthrene (S₂), phenanthridine (N₁), phenanthroline (N₂),        phenazine (N₂).

The above groups, whether alone or part of another substituent, maythemselves optionally be substituted with one or more groups selectedfrom themselves and the additional substituents listed below.

Halo: —F, —Cl, —Br, and —I.

Hydroxy: —OH.

Ether: —OR, wherein R is an ether substituent, for example, a C₁₋₇ alkylgroup (also referred to as a C₁₋₇ alkoxy group, discussed below), aC₃₋₂₀ heterocyclyl group (also referred to as a C₃₋₂₀ heterocyclyloxygroup), or a C₅₋₂₀ aryl group (also referred to as a C₅₋₂₀ aryloxygroup), preferably a C₁₋₇alkyl group.

Alkoxy: —OR, wherein R is an alkyl group, for example, a C₁₋₇ alkylgroup. Examples of C₁₋₇ alkoxy groups include, but are not limited to,—OMe (methoxy), —OEt (ethoxy), —O(nPr) (n-propoxy), —O(iPr)(isopropoxy), —O(nBu) (n-butoxy), —O(sBu) (sec-butoxy), —O(iBu)(isobutoxy), and —O(tBu) (tert-butoxy).

Acetal: —CH(OR¹)(OR²), wherein R¹ and R² are independently acetalsubstituents, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group, or, in thecase of a “cyclic” acetal group, R¹ and R², taken together with the twooxygen atoms to which they are attached, and the carbon atoms to whichthey are attached, form a heterocyclic ring having from 4 to 8 ringatoms. Examples of acetal groups include, but are not limited to,—CH(OMe)₂, —CH(OEt)₂, and —CH(OMe)(OEt).

Hemiacetal: —CH(OH)(OR¹), wherein R¹ is a hemiacetal substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group. Examples of hemiacetal groupsinclude, but are not limited to, —CH(OH)(OMe) and —CH(OH)(OEt).

Ketal: —CR(OR¹)(OR²), where R¹ and R² are as defined for acetals, and Ris a ketal substituent other than hydrogen, for example, a C₁₋₇ alkylgroup, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably aC₁₋₇ alkyl group. Examples ketal groups include, but are not limited to,—C(Me)(OMe)₂, —C(Me)(OEt)₂, —C(Me)(OMe)(OEt), —C(Et)(OMe)₂,—C(Et)(OEt)₂, and —C(Et)(OMe)(OEt).

Hemiketal: —CR(OH)(OR¹), where R¹ is as defined for hemiacetals, and Ris a hemiketal substituent other than hydrogen, for example, a C₁₋₇alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group,preferably a C₁₋₇ alkyl group. Examples of hemiacetal groups include,but are not limited to, —C(Me)(OH)(OMe), —C(Et)(OH)(OMe),—C(Me)(OH)(OEt), and —C(Et)(OH)(OEt).

Oxo (keto, -one): ═O.

Thione (thioketone): ═S.

Imino (imine): ═NR, wherein R is an imino substituent, for example,hydrogen, C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably hydrogen or a C₁₋₇ alkyl group. Examples of estergroups include, but are not limited to, ═NH, ═NMe, ═NEt, and ═NPh.

Formyl (carbaldehyde, carboxaldehyde): —C(═O)H.

Acyl (keto): —C(═O)R, wherein R is an acyl substituent, for example, aC₁₋₇ alkyl group (also referred to as C₁₋₇alkylacyl or C₁₋₇alkanoyl), aC₃₋₂₀ heterocyclyl group (also referred to as C₃₋₂₀ heterocyclylacyl),or a C₅₋₂₀ aryl group (also referred to as C₅₋₂₀ arylacyl), preferably aC₁₋₇ alkyl group. Examples of acyl groups include, but are not limitedto, —C(═O)CH₃ (acetyl), —C(═O)CH₂CH₃ (propionyl), —C(═O)C(CH₃)₃(t-butyryl), and —C(═O)Ph (benzoyl, phenone).

Carboxy (carboxylic acid): —C(═O)OH.

Thiocarboxy (thiocarboxylic acid): —C(═S)SH.

Thiolocarboxy (thiolocarboxylic acid): —C(═O)SH.

Thionocarboxy (thionocarboxylic acid): —C(═S)OH.

Imidic acid: —C(═NH)OH.

Hydroxamic acid: —C(═NOH)OH.

Ester (carboxylate, carboxylic acid ester, oxycarbonyl): —C(═O)OR,wherein R is an ester substituent, for example, a C₁₋₇ alkyl group, aC₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkylgroup. Examples of ester groups include, but are not limited to,—C(═O)OCH₃, —C(═O)OCH₂CH₃, —C(═O)OC(CH₃)₃, and —C(═O)OPh.

Acyloxy (reverse ester): —OC(═O)R, wherein R is an acyloxy substituent,for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀aryl group, preferably a C₁₋₇ alkyl group. Examples of acyloxy groupsinclude, but are not limited to, —OC(═O)CH₃ (acetoxy), —OC(═O)CH₂CH₃,—OC(═O)C(CH₃)₃, —OC(═O)Ph, and —OC(═O)CH₂Ph.

Oxycarboyloxy: —OC(═O)OR, wherein R is an ester substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group. Examples of ester groups include,but are not limited to, —OC(═O)OCH₃, —OC(═O)OCH₂CH₃, —OC(═O)OC(CH₃)₃,and —OC(═O)OPh.

Amino: —NR¹R², wherein R¹ and R² are independently amino substituents,for example, hydrogen, a C₁₋₇ alkyl group (also referred to as C₁₋₇alkylamino or di-C₁₋₇ alkylamino), a C₃₋₂₀ heterocyclyl group, or aC₅₋₂₀ aryl group, preferably H or a C₁₋₇ alkyl group, or, in the case ofa “cyclic” amino group, R¹ and R², taken together with the nitrogen atomto which they are attached, form a heterocyclic ring having from 4 to 8ring atoms. Amino groups may be primary (—NH₂), secondary (—NHR¹), ortertiary (—NHR¹R²), and in cationic form, may be quaternary (—⁺NR¹R²R³).Examples of amino groups include, but are not limited to, —NH₂, —NHCH₃,—NHC(CH₃)₂, —N(CH₃)₂, —N(CH₂CH₃)₂, and —NHPh. Examples of cyclic aminogroups include, but are not limited to, aziridino, azetidino,pyrrolidino, piperidino, piperazino, morpholino, and thiomorpholino.

Amido (carbamoyl, carbamyl, aminocarbonyl, carboxamide): —C(═O)NR¹R²,wherein R¹ and R² are independently amino substituents, as defined foramino groups. Examples of amido groups include, but are not limited to,—C(═O)NH₂, —C(═O)NHCH₃, —C(═O)N(CH₃)₂, —C(═O)NHCH₂CH₃, and—C(═O)N(CH₂CH₃)₂, as well as amido groups in which R¹ and R², togetherwith the nitrogen atom to which they are attached, form a heterocyclicstructure as in, for example, piperidinocarbonyl, morpholinocarbonyl,thiomorpholinocarbonyl, and piperazinocarbonyl.

Thioamido (thiocarbamyl): —C(═S)NR¹R², wherein R¹ and R² areindependently amino substituents, as defined for amino groups. Examplesof amido groups include, but are not limited to, —C(═S)NH₂, —C(═S)NHCH₃,—C(═S)N(CH₃)₂, and —C(═S)NHCH₂CH₃.

Acylamido (acylamino): —NR¹C(═O)R², wherein R¹ is an amide substituent,for example, hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group,or a C₅₋₂₀ aryl group, preferably hydrogen or a C₁₋₇ alkyl group, and R²is an acyl substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀heterocyclyl group, or a C₅₋₂₀aryl group, preferably hydrogen or a C₁₋₇alkyl group. Examples of acylamide groups include, but are not limitedto, —NHC(═O)CH₃, —NHC(═O)CH₂CH₃, and —NHC(═O)Ph. R¹ and R² may togetherform a cyclic structure, as in, for example, succinimidyl, maleimidyl,and phthalimidyl:

Aminocarbonyloxy: —OC(═O)NR¹R², wherein R¹ and R² are independentlyamino substituents, as defined for amino groups. Examples ofaminocarbonyloxy groups include, but are not limited to, —OC(═O)NH₂,—OC(═O)NHMe, —OC(═O)NMe₂, and —OC(═O)NEt₂.

Ureido: —N(R¹)CONR²R³ wherein R² and R³ are independently aminosubstituents, as defined for amino groups, and R¹ is a ureidosubstituent, for example, hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀heterocyclyl group, or a C₅₋₂₀ aryl group, preferably hydrogen or a C₁₋₇alkyl group. Examples of ureido groups include, but are not limited to,—NHCONH₂, —NHCONHMe, —NHCONHEt, —NHCONMe₂, —NHCONEt₂, —NMeCONH₂,—NMeCONHMe, —NMeCONHEt, —NMeCONMe₂, and —NMeCONEt₂.

Guanidino: —NH—C(═NH)NH₂.

Tetrazolyl: a five membered aromatic ring having four nitrogen atoms andone carbon atom,

Imino: ═NR, wherein R is an imino substituent, for example, for example,hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀aryl group, preferably H or a C₁₋₇alkyl group. Examples of imino groupsinclude, but are not limited to, ═NH, ═NMe, and ═NEt.

Amidine (amidino): —C(═NR)NR₂, wherein each R is an amidine substituent,for example, hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group,or a C₅₋₂₀ aryl group, preferably H or a C₁₋₇ alkyl group. Examples ofamidine groups include, but are not limited to, —C(═NH)NH₂, —C(═NH)NMe₂,and —C(═NMe)NMe₂.

Nitro: —NO₂.

Nitroso: —NO.

Azido: —N₃.

Cyano (nitrile, carbonitrile): —CN.

Isocyano: —NC.

Cyanato: —OCN.

Isocyanato: —NCO.

Thiocyano (thiocyanato): —SCN.

Isothiocyano (isothiocyanato): —NCS.

Sulfhydryl (thiol, mercapto): —SH.

Thioether (sulfide): —SR, wherein R is a thioether substituent, forexample, a C₁₋₇ alkyl group (also referred to as a C₁₋₇alkylthio group),a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇alkyl group. Examples of C₁₋₇ alkylthio groups include, but are notlimited to, —SCH₃ and —SCH₂CH₃.

Disulfide: —SS—R, wherein R is a disulfide substituent, for example, aC₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group,preferably a C₁₋₇ alkyl group (also referred to herein as C₁₋₇ alkyldisulfide). Examples of C₁₋₇ alkyl disulfide groups include, but are notlimited to, —SSCH₃ and —SSCH₂CH₃.

Sulfine (sulfinyl, sulfoxide): —S(═O)R, wherein R is a sulfinesubstituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples ofsulfine groups include, but are not limited to, —S(═O)CH₃ and—S(═O)CH₂CH₃.

Sulfone (sulfonyl): —S(═O)₂R, wherein R is a sulfone substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group, including, for example, afluorinated or perfluorinated C₁₋₇ alkyl group. Examples of sulfonegroups include, but are not limited to, —S(═O)₂CH₃ (methanesulfonyl,mesyl), —S(═O)₂CF₃ (triflyl), —S(═O)₂CH₂CH₃ (esyl), —S(═O)₂C₄F₉(nonaflyl), —S(═O)₂CH₂CF₃ (tresyl), —S(═O)₂CH₂CH₂NH₂ (tauryl), —S(═O)₂Ph(phenylsulfonyl, besyl), 4-methylphenylsulfonyl (tosyl),4-chlorophenylsulfonyl (closyl), 4-bromophenylsulfonyl (brosyl),4-nitrophenyl (nosyl), 2-naphthalenesulfonate (napsyl), and5-dimethylamino-naphthalen-1-ylsulfonate (dansyl).

Sulfinic acid (sulfino): —S(═O)OH, —SO₂H.

Sulfonic acid (sulfo): —S(═O)₂OH, —SO₃H.

Sulfinate (sulfinic acid ester): —S(═O)OR; wherein R is a sulfinatesubstituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples ofsulfinate groups include, but are not limited to, —S(═O)OCH₃(methoxysulfinyl; methyl sulfinate) and —S(═O)OCH₂CH₃ (ethoxysulfinyl;ethyl sulfinate).

Sulfonate (sulfonic acid ester): —S(═O)₂OR, wherein R is a sulfonatesubstituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples ofsulfonate groups include, but are not limited to, —S(═O)₂OCH₃(methoxysulfonyl; methyl sulfonate) and —S(═O)₂OCH₂CH₃ (ethoxysulfonyl;ethyl sulfonate).

Sulfinyloxy: —OS(═O)R, wherein R is a sulfinyloxy substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₃₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group. Examples of sulfinyloxy groupsinclude, but are not limited to, —OS(═O)CH₃ and —OS(═O)CH₂CH₃.

Sulfonyloxy: —OS(═O)₂R, wherein R is a sulfonyloxy substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group. Examples of sulfonyloxy groupsinclude, but are not limited to, —OS(═O)₂CH₃ (mesylate) and—OS(═O)₂CH₂CH₃ (esylate).

Sulfate: —OS(═O)₂OR; wherein R is a sulfate substituent, for example, aC₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group,preferably a C₁₋₇ alkyl group. Examples of sulfate groups include, butare not limited to, —OS(═O)₂OCH₃ and —SO(═O)₂OCH₂CH₃.

Sulfamyl (sulfamoyl; sulfinic acid amide; sulfinamide): —S(═O)NR¹R²,wherein R¹ and R² are independently amino substituents, as defined foramino groups. Examples of sulfamyl groups include, but are not limitedto, —S(═O)NH₂, —S(═O)NH(CH₃), —S(═O)N(CH₃)₂, —S(═O)NH(CH₂CH₃),—S(═O)N(CH₂CH₃)₂, and —S(═O)NHPh.

Sulfonamido (sulfinamoyl; sulfonic acid amide; sulfonamide):—S(═O)₂NR¹R², wherein R¹ and R² are independently amino substituents, asdefined for amino groups. Examples of sulfonamido groups include, butare not limited to, —S(═O)₂NH₂, —S(═O)₂NH(CH₃), —S(═O)₂N(CH₃)₂,—S(═O)₂NH(CH₂CH₃), —S(═O)₂N(CH₂CH₃)₂, and —S(═O)₂NHPh.

Sulfamino: —NR¹S(═O)₂OH, wherein R¹ is an amino substituent, as definedfor amino groups. Examples of sulfamino groups include, but are notlimited to, —NHS(═O)₂OH and —N(CH₃)S(═O)₂OH.

Sulfonamino: —NR¹S(═O)₂R, wherein R¹ is an amino substituent, as definedfor amino groups, and R is a sulfonamino substituent, for example, aC₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group,preferably a C₁₋₇ alkyl group. Examples of sulfonamino groups include,but are not limited to, —NHS(═O)₂CH₃ and —N(CH₃)S(═O)₂C₆H₅.

Sulfinamino: —NR¹S(═O)R, wherein R¹ is an amino substituent, as definedfor amino groups, and R is a sulfinamino substituent, for example, aC₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₃₋₂₀ aryl group,preferably a C₁₋₇ alkyl group. Examples of sulfinamino groups include,but are not limited to, —NHS(═O)CH₃ and —N(CH₃)S(═O)C₆H₅.

Phosphino (phosphine): —PR₂, wherein R is a phosphino substituent, forexample, —H, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀aryl group, preferably —H, a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group.Examples of phosphino groups include, but are not limited to, —PH₂,—P(CH₃)₂, —P(CH₂CH₃)₂, —P(t-Bu)₂, and —P(Ph)₂.

Phospho: —P(═O)₂.

Phosphinyl (phosphine oxide): —P(═O)R₂, wherein R is a phosphinylsubstituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₃₋₂₀ aryl group, preferably a C₁₋₇ alkyl group or a C₅₋₂₀aryl group. Examples of phosphinyl groups include, but are not limitedto, —P(═O)(CH₃)₂, —P(═O)(CH₂CH₃)₂, —P(═O)(t-Bu)₂, and —P(═O)(Ph)₂.

Phosphonic acid (phosphono): —P(═O)(OH)₂.

Phosphonate (phosphono ester): —P(═O)(OR)₂, where R is a phosphonatesubstituent, for example, —H, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably —H, a C₁₋₇ alkyl group, or aC₅₋₂₀ aryl group. Examples of phosphonate groups include, but are notlimited to, —P(═O)(OCH₃)₂, —P(═O)(OCH₂CH₃)₂, —P(═O)(O-t-Bu)₂, and—P(═O)(OPh)₂.

Phosphoric acid (phosphonooxy): —OP(═O)(OH)₂.

Phosphate (phosphonooxy ester): —OP(═O)(OR)₂, where R is a phosphatesubstituent, for example, —H, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably —H, a C₁₋₇ alkyl group, or aC₅₋₂₀ aryl group. Examples of phosphate groups include, but are notlimited to, —OP(═O)(OCH₃)₂, —OP(═O)(OCH₂CH₃)₂, —OP(═O)(O-t-Bu)₂, and—OP(═O)(OPh)₂.

Phosphorous acid: —OP(OH)₂.

Phosphite: —OP(OR)₂, where R is a phosphite substituent, for example,—H, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably —H, a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group.Examples of phosphite groups include, but are not limited to,—OP(OCH₃)₂, —OP(OCH₂CH₃)₂, —OP(O-t-Bu)₂, and —OP(OPh)₂.

Phosphoramidite: —OP(OR¹)—NR₂ ², where R¹ and R² are phosphoramiditesubstituents, for example, —H, a (optionally substituted) C₁₋₇ alkylgroup, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably —H,a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group. Examples of phosphoramiditegroups include, but are not limited to, —OP(OCH₂CH₃)—N(CH₃)₂,—OP(OCH₂CH₃)—N(i-Pr)₂, and —OP(OCH₂CH₂CN)—N(i-Pr)₂.

Phosphoramidate: —OP(═O)(OR¹)—NR₂ ², where R¹ and R² are phosphoramidatesubstituents, for example, —H, a (optionally substituted) C₁₋₇ alkylgroup, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably —H,a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group. Examples of phosphoramidategroups include, but are not limited to, —OP(═O)(OCH₂CH₃)—N(CH₃)₂,—OP(═O)(OCH₂CH₃)—N(i-Pr)₂, and —OP(═O)(OCH₂CH₂CN)—N(i-Pr)₂.

Bisoxyalkylene: —O—(CH₂)_(n)—O—, where n=1-3, which bonds to adjacentatoms. Examples of bisoxyalkylene groups include, but are not limitedto, —O—CH₂—O—.

Alkylene

C₃₋₁₂ alkylene: The term “C₃₋₁₂ alkylene”, as used herein, pertains to abidentate moiety obtained by removing two hydrogen atoms, either bothfrom the same carbon atom, or one from each of two different carbonatoms, of a hydrocarbon compound having from 3 to 12 carbon atoms(unless otherwise specified), which may be aliphatic or alicyclic, andwhich may be saturated, partially unsaturated, or fully unsaturated.Thus, the term “alkylene” includes the sub-classes alkenylene,alkynylene, cycloalkylene, etc., discussed below.

Examples of linear saturated C₃₋₁₂ alkylene groups include, but are notlimited to, —(CH₂)_(n)—where n is an integer from 3 to 12, for example,—CH₂CH₂CH₂— (propylene), —CH₂CH₂CH₂CH₂— (butylene), —CH₂CH₂CH₂CH₂CH₂—(pentylene) and —CH₂CH₂CH₂CH₂CH₂CH₂CH₂— (heptylene).

Examples of branched saturated C₃₋₁₂ alkylene groups include, but arenot limited to, —CH(CH₃)CH₂—, —CH(CH₃)CH₂CH₂—, —CH(CH₃)CH₂CH₂CH₂—,—CH₂CH(CH₃)CH₂—, —CH₂CH(CH₃)CH₂CH₂—, —CH(CH₂CH₃)—, —CH(CH₂CH₃)CH₂—, and—CH₂CH(CH₂CH₃)CH₂—.

Examples of linear partially unsaturated C₃₋₁₂ alkylene groups (C₃₋₁₂alkenylene, and alkynylene groups) include, but are not limited to,—CH═CH—CH₂—, —CH₂—CH═CH₂—, —CH═CH—CH₂—CH₂—, —CH═CH—CH₂—CH₂—CH₂—,—CH═CH—CH═CH—, —CH═CH—CH═CH—CH₂—, —CH═CH—CH═CH—CH₂—CH₂—,—CH═CH—CH₂—CH═CH—, —CH═CH—CH₂—CH₂—CH═CH—, and —CH₂—C≡C—CH₂—.

Examples of branched partially unsaturated C₃₋₁₂ alkylene groups (C₃₋₁₂alkenylene and alkynylene groups) include, but are not limited to,—C(CH₃)═CH—, —C(CH₃)═CH—CH₂—, —CH═CH—CH(CH₃)— and —C≡C—CH(CH₃)—.

Examples of alicyclic saturated C₃₋₁₂ alkylene groups (C₃₋₁₂cycloalkylenes) include, but are not limited to, cyclopentylene (e.g.cyclopent-1,3-ylene), and cyclohexylene (e.g. cyclohex-1,4-ylene).

Examples of alicyclic partially unsaturated C₃₋₁₂ alkylene groups (C₃₋₁₂cycloalkylenes) include, but are not limited to, cyclopentenylene (e.g.4-cyclopenten-1,3-ylene), cyclohexenylene (e.g. 2-cyclohexen-1,4-ylene;3-cyclohexen-1,2-ylene; 2,5-cyclohexadien-1,4-ylene).

Pharmaceutically Acceptable Cations

Examples of pharmaceutically acceptable monovalent and divalent cationsare discussed in Berge, et al., J. Pharm. Sci., 66, 1-19 (1977), whichis incorporated herein by reference.

The pharmaceutically acceptable cation may be inorganic or organic.

Examples of pharmaceutically acceptable monovalent inorganic cationsinclude, but are not limited to, alkali metal ions such as Na⁺ and K⁺.Examples of pharmaceutically acceptable divalent inorganic cationsinclude, but are not limited to, alkaline earth cations such as Ca²⁺ andMg²⁺. Examples of pharmaceutically acceptable organic cations include,but are not limited to, ammonium ion (i.e. NH₄ ⁺) and substitutedammonium ions (e.g. NH₃R⁺, NH₂R₂ ⁺, NHR₃ ⁺, NR₄ ⁺). Examples of somesuitable substituted ammonium ions are those derived from: ethylamine,diethylamine, dicyclohexylamine, triethylamine, butylamine,ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine,phenylbenzylamine, choline, meglumine, and tromethamine, as well asamino acids, such as lysine and arginine. An example of a commonquaternary ammonium ion is N(CH₃)₄ ⁺.

Proliferative Diseases

One of ordinary skill in the art is readily able to determine whether ornot a candidate compound treats a proliferative condition for anyparticular cell type. For example, assays which may conveniently be usedto assess the activity offered by a particular compound are described inthe examples below.

The term “proliferative disease” pertains to an unwanted or uncontrolledcellular proliferation of excessive or abnormal cells which isundesired, such as, neoplastic or hyperplastic growth, whether in vitroor in vivo.

Examples of proliferative conditions include, but are not limited to,benign, pre-malignant, and malignant cellular proliferation, includingbut not limited to, neoplasms and tumours (e.g. histocytoma, glioma,astrocyoma, osteoma), cancers (e.g. lung cancer, small cell lung cancer,gastrointestinal cancer, bowel cancer, colon cancer, breast carinoma,ovarian carcinoma, prostate cancer, testicular cancer, liver cancer,kidney cancer, bladder cancer, pancreas cancer, brain cancer, sarcoma,osteosarcoma, Kaposi's sarcoma, melanoma), leukemias, psoriasis, bonediseases, fibroproliferative disorders (e.g. of connective tissues), andatherosclerosis.

Any type of cell may be treated, including but not limited to, lung,gastrointestinal (including, e.g. bowel, colon), breast (mammary),ovarian, prostate, liver (hepatic), kidney (renal), bladder, pancreas,brain, and skin.

Methods of Treatment

As described above, the present invention provide the use of a compoundof formula I in a method of therapy. Also provided is a method oftreatment, comprising administering to a subject in need of treatment atherapeutically-effective amount of a compound of formula I, preferablyin the form of a pharmaceutical composition. The term “therapeuticallyeffective amount” is an amount sufficient to show benefit to a patient.Such benefit may be at least amelioration of at least one symptom. Theactual amount administered, and rate and time-course of administration,will depend on the nature and severity of what is being treated.Prescription of treatment, e.g. decisions on dosage, is within theresponsibility of general practitioners and other medical doctors.

A compound may be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated. Examples of treatments and therapies include,but are not limited to, chemotherapy (the administration of activeagents, including, e.g. drugs; surgery; and radiation therapy.

Pharmaceutical compositions according to the present invention, and foruse in accordance with the present invention, may comprise, in additionto the active ingredient, i.e. a compound of formula I, apharmaceutically acceptable excipient, carrier, buffer, stabiliser orother materials well known to those skilled in the art. Such materialsshould be non-toxic and should not interfere with the efficacy of theactive ingredient. The precise nature of the carrier or other materialwill depend on the route of administration, which may be oral, or byinjection, e.g. cutaneous, subcutaneous, or intravenous.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may comprise a solid carrier oran adjuvant. Liquid pharmaceutical compositions generally comprise aliquid carrier such as water, petroleum, animal or vegetable oils,mineral oil or synthetic oil. Physiological saline solution, dextrose orother saccharide solution or glycols such as ethylene glycol, propyleneglycol or polyethylene glycol may be included. A capsule may comprise asolid carrier such a gelatin.

For intravenous, cutaneous or subcutaneous injection, or injection atthe site of affliction, the active ingredient will be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection,Lactated Ringer's Injection. Preservatives, stabilisers, buffers,antioxidants and/or other additives may be included, as required.

Includes Other Forms

Unless otherwise specified, included in the above are the well knownionic, salt, solvate, and protected forms of these substituents. Forexample, a reference to carboxylic acid (—COOH) also includes theanionic (carboxylate) form (—COO), a salt or solvate thereof, as well asconventional protected forms. Similarly, a reference to an amino groupincludes the protonated form (—N⁺HR¹R²), a salt or solvate of the aminogroup, for example, a hydrochloride salt, as well as conventionalprotected forms of an amino group. Similarly, a reference to a hydroxylgroup also includes the anionic form (—O⁻), a salt or solvate thereof,as well as conventional protected forms.

In particular, a reference to group (—SO_(z)M) also includes the anionicform (—SO_(z) ⁻), or solvate thereof, as well as conventional protectedforms.

Isomers and Solvates

Certain compounds may exist in one or more particular geometric,optical, enantiomeric, diasteriomeric, epimeric, atropic,stereoisomeric, tautomeric, conformational, or anomeric forms, includingbut not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, andr-forms; endo- and exo-forms; R—, S—, and meso-forms; D- and L-forms; d-and l-forms; (+) and (−) forms; keto-, enol-, and enolate-forms; syn-and anti-forms; synclinal- and anticlinal-forms; α- and β-forms; axialand equatorial forms; boat-, chair-, twist-, envelope-, andhalfchair-forms; and combinations thereof, hereinafter collectivelyreferred to as “isomers” (or “isomeric forms”).

In some embodiments, compounds of the present invention have thefollowing stereochemistry at the C11 position:

Note that, except as discussed below for tautomeric forms, specificallyexcluded from the term “isomers”, as used herein, are structural (orconstitutional) isomers (i.e. isomers which differ in the connectionsbetween atoms rather than merely by the position of atoms in space). Forexample, a reference to a methoxy group, —OCH₃, is not to be construedas a reference to its structural isomer, a hydroxymethyl group, —CH₂OH.Similarly, a reference to ortho-chlorophenyl is not to be construed as areference to its structural isomer, meta-chlorophenyl. However, areference to a class of structures may well include structurallyisomeric forms falling within that class (e.g. C₁₋₇ alkyl includesn-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl;methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).

The above exclusion does not pertain to tautomeric forms, for example,keto-, enol-, and enolate-forms, as in, for example, the followingtautomeric pairs: keto/enol (illustrated below), imine/enamine,amide/imino alcohol, amidine/amidine, nitroso/oxime,thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro.

Note that specifically included in the term “isomer” are compounds withone or more isotopic substitutions. For example, H may be in anyisotopic form, including ¹H, ²H (D), and ³H (T); C may be in anyisotopic form, including ¹²C, ¹³C, and ¹⁴C; O may be in any isotopicform, including ¹⁶O and ¹⁸O; and the like.

Unless otherwise specified, a reference to a particular compoundincludes all such isomeric forms, including (wholly or partially)racemic and other mixtures thereof. Methods for the preparation (e.g.asymmetric synthesis) and separation (e.g. fractional crystallisationand chromatographic means) of such isomeric forms are either known inthe art or are readily obtained by adapting the methods taught herein,or known methods, in a known manner.

It may be convenient or desirable to prepare, purify, and/or handle acorresponding solvate of the active compound. The term “solvate” is usedherein in the conventional sense to refer to a complex of solute (e.g.active compound, salt of active compound) and solvent. If the solvent iswater, the solvate may be conveniently referred to as a hydrate, forexample, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.

General Synthetic Routes

The synthesis of PBD compounds is extensively discussed in WO 00/12508,which discussion is incorporated herein by reference.

As discussed in that patent application, a key step in a preferred routeto PBDs is a cyclisation to produce the B-ring, involving generation ofan aldehyde (or functional equivalent thereof) at what will be the11-position, and attack thereon by the Pro-N10-nitrogen:

wherein the substituents are as defined above, R⁸ represents the link(—X—R″—X—) to the other PBD moiety, R¹⁰ is a nitrogen protecting groupand R¹² is R² or a precursor thereof. The “masked aldehyde” —CPQ may bean acetal or thioacetal, in which case the cyclisation involvesunmasking. Alternatively, it may be an alcohol —CHOH, in which case thereaction involves oxidation, e.g. by means of TPAP, TEMPO or DMSO (Swernoxidation).

The masked aldehyde compound can be produced by condensing acorresponding 2,4-substituted pyrrolidine with a 2-nitrobenzoic acid:

The nitro group can then be reduced to —NH₂ and protected by reactionwith a suitable agent, e.g. a chloroformate, which provides theremovable nitrogen protecting group in the compound of formula IV.

A process involving the oxidation-cyclization procedure is illustratedin scheme 1 (an alternative type of cyclisation will be described laterwith reference to scheme 2).

Exposure of the alcohol (B) (in which the Pro-N10-nitrogen is generallyprotected as carbamate) to tetrapropylammonium perruthenate(TPAP)/N-methylmorpholine N-oxide (NMO) over A4 sieves results inoxidation accompanied by spontaneous B-ring closure to afford thedesired product IV. The TPAP/NMO oxidation procedure is found to beparticularly convenient for small scale reactions while the use ofDMSO-based oxidation methods, particularly Swern oxidation, provessuperior for larger scale work (e.g. >1 g). A particularly preferredoxidising agent is (diacetoxyiodo)benzene (1.1 eq) and TEMPO (0.1 eq)dissolved in CH₂Cl₂.

The uncyclized alcohol (B) may be prepared by the reaction of a nitrogenprotection reagent of formula D, which is preferably a chloroformate oracid chloride, to a solution of the amino alcohol C, generally insolution, generally in the presence of a base such as pyridine(preferably 2 equivalents) at a moderate temperature (e.g. at 0° C.).Under these conditions little or no O-acylation is usually observed.

The key amino alcohol C may be prepared by reduction of thecorresponding nitro compound E, by choosing a method which will leavethe rest of the molecule intact. Treatment of E with tin (II) chloridein a suitable solvent, e.g. refluxing methanol, generally affords, afterthe removal of the tin salts, the desired product in high yield.

Exposure of E to hydrazine/Raney nickel avoids the production of tinsalts and may result in a higher yield of C, although this method isless compatible with the range of possible C and A-ring substituents.For instance, if there is C-ring unsaturation (either in the ringitself, or in R₂ or R₃), this technique may be unsuitable. Anothersuitable means of reduction would be catalytic hydrogenation usingpalladium on carbon as a catalyst.

The nitro compound of formula E may be prepared by coupling theappropriate o-nitrobenzoyl chloride to a compound of formula F, e.g. inthe presence of K₂CO₃ at −25° C. under a N₂ atmosphere. Compounds offormula F can be readily prepared, for example by olefination of theketone derived from L-trans-hydroxy proline. The ketone intermediate canalso be exploited by conversion to the enol triflate for use inpalladium mediated coupling reactions.

The o-nitrobenzoyl chloride is synthesised from the o-nitrobenzoic acid(or alkyl ester after hydrolysis) of formula G, which itself is preparedfrom the vanillic acid (or alkyl ester) derivative H. Many of these arecommercially available and some are disclosed in Althuis, T. H. andHess, H. J., J. Medicinal Chem., 20(1), 146-266 (1977).

Alternative Cyclisation (Scheme 2)

In scheme 1, the final or penultimate step was an oxidative cyclisation.An alternative, using thioacetal coupling, is shown in scheme 2.Mercury-mediated unmasking causes cyclisation to the protected PBDcompound IV.

The thioacetal compound may be prepared as shown in scheme 2: thethioacetal protected C-ring [prepared via a literature method: Langley,D. R. & Thurston, D. E., J. Organic Chemistry, 52, 91-97 (1987)] iscoupled to the o-nitrobenzoic acid (or alkyl ester after hydrolysis) (G)using a literature procedure. The resulting nitro compound cannot bereduced by hydrogenation, because of the thioacetal group, so thetin(II) chloride method is used to afford the amine. This is thenN-protected, e.g., by reaction with a chloroformate or acid chloride,such as 2,2,2-trichloroethylchloroformate.

Acetal-containing C-rings can be used as an alternative in this type ofroute with deprotection involving other methods, including the use ofacidic conditions.

Dimer Synthesis (Scheme 3)

PBD dimers, as in the present invention, may be synthesized using thestrategy developed for the synthesis of the protected PBD monomers. Thesynthesis routes illustrated in scheme 3 show compounds when the dimerlinkage is of the formula —O—(CH₂)_(n)—O—, and may be readily modifiedfor other dimer linkages. The step of dimer formation is normallycarried out to form a bis(nitro acid) G′. This compound can then betreated as compound G in either scheme 1 or scheme 2 above.

The bis(nitro acid) G′ may be obtained by nitrating (e.g. using 70%nitric acid) the bis(carboxylic acid). This can be synthesised byalkylation of two equivalents of the relevant benzoic acid with theappropriate diiodoalkane under basic conditions. Many benzoic acids arecommercially available and others can be synthesised by conventionalmethods. Alternatively, the relevant benzoic acid esters can be joinedtogether by a Mitsunobu etherification with an appropriate alkanediol,followed by nitration, and then hydrolysis (not illustrated).

An alternative synthesis of the bis(nitro acid) involves oxidation ofthe bis(nitro aldehyde), e.g. with potassium permanganate. This can beobtained in turn by direct nitration of the bis(aldehyde), e.g. with 70%HNO₃. Finally, the bis(aldehyde) can be obtained via the Mitsunobuetherification of two equivalents of the benzoic aldehyde with theappropriate alkanediol.

Alternative Routes to PBDs

Alternative methods of synthesising N10 protected PBDs are disclosed WO2005/023814, which is incorporated herein, and which describes the useof isocyanate intermediates.

Introduction of C2 Aryl Substituent: Introducing Leaving Group at C2

The C2 aryl substituent may be in place in compounds of formula IV, bystarting with the appropriate material, in which case the N10 may bedeprotected to yield the desired compound (see below). Alternatively,the following method can be used where R¹² is a protected hydroxy group.Following cyclisation to form the B-ring, the C11-alcohol IV is thenpreferably re-protected, by conventional means to provide IIIb. Forexample, if R¹¹ is TBS, the protection can take place by reacting IVwith TBSOTf and 2,6-lutidine. Cleavage of the C2-protecting group fromIIIb then provides the C2 alcohol. For example, where the C2 protectinggroup (R¹⁴) is acyl, this deprotection may be performed by addition ofan aqueous solution of K₂CO₃.

This reprotection at the C11 position and deprotection of the C2 alcoholallows subsequent reaction of selectively the C2 alcohol positionleaving the C11 position unaffected. The C2-alcohol may then be oxidizedto the ketone IIIb. Preferably this oxidation is performed under Swernconditions, in good yield. However, other oxidation methods involvingTPAP or the Dess Martin reagent also provide the ketone in good yield.

R″¹² in the compound of formula II may be —OSO₂CH₃, —OSO₂(C_(n)F_(2n+1))where n=0, 1 or 4, or —OSO₂R^(S), in which case the conversion from IIIbmay be achieved by treatment with the appropriate anhydride. Forexample, if R″¹² is trfilate that reaction with trifluoromethanesulfonicanhydride in DCM in the presence of pyridine.

R″¹² in the compound of formula II may also be —I or —Br, in which casethe conversion from IIIb may be achieved by reaction with hydrazine andiodine or bromine respectively.

R″¹² in the compound of formula II may also be —Cl, where the conversionfrom IIIb may be achieved by reaction with a phosphorous oxychloride(e.g. POCl₃).

Introduction of C2 Aryl Substituent: Replacement of Leaving Group

This compound of formula II may be reacted under a variety of conditionsto yield PBD precursor molecules with pendant groups coupled at the C2position Ic.

In particular, the use of palladium catalysed coupling is preferred,such as Suzuki, Stille and Heck coupling. The palladium catalyst may beany suitable catalyst, for example Pd(PPh₃)₄, Pd(OCOCH₃)₂, PdCl₂,Pd(dba)₃. The compounds which are coupled may be any suitable reactant,e.g. for Heck, alkenes with an sp²H; for Stille, organostannanes; andfor Suzuki, organoboron derivatives.

In a preferred aspect of the invention, the coupling may be performedunder microwave conditions. Typically, the palladium catalyst, such asPd(PPh₃)₄, is solid supported, for example on polystyrene, to facilitatework-up and allow potential recycling of catalyst. Unreacted boronicacid can be sequestered following complete consumption of triflate usingPS-DEAM, with a phase separator cartridge being used to isolate thecoupling product. Such a method allows for the parallel synthesis ofmore than one (e.g. up to 10, or 30) compound at the same time.

The imine bond in the compound of formula Ic can be unprotected bystandard methods to yield the unprotected compound Ib (which may be inits carbinolamine or carboinolamine ether form, depending on thesolvents used). For example if R¹⁹ is Alloc, then the deprotection iscarried using palladium to remove the N10 protecting group, followed bythe elimination of water. If R¹⁹ is Troc, then the deprotection iscarried out using a Cd/Pb couple to yield the compound of formula Ib.

Reference is also made to the synthesis discussion and examples in WO2004/043963 and WO 2005/085251, which are herein incorporated byreference.

Conversion to Sulphur Containing Form

The conversion of compounds of formula Ib to those of the presentinvention may be carried out by the addition of the appropriatebisulphite salt or sulphinate salt, followed by an appropriatepurification step. Further methods are described in GB 2 053 894, whichis herein incorporated by reference.

Further Preferences

It is preferred that X is O.

It is preferred that R″ represents a linear saturated C₃₋₁₂ alkylenegroup, and more preferably an alkylene group having 3, 5, 7, 8, 9, 10,11 or 12 carbon atoms. Of these C₃ and C₅ linear saturated alkylenegroups are preferred.

R⁹ is preferably H.

R⁶ is preferably selected from H, OH, OR, SH, NH₂, nitro and halo, andis more preferably H or halo, and most preferably is H.

R⁷ is preferably selected from H, OH, OR, SH, SR, NH₂, NHR, NRR′, andhalo, and more preferably independently selected from H, OH and OR,where R is preferably selected from optionally substituted C₁₋₇ alkyl,C₃₋₁₀ heterocyclyl and C₃₋₁₀ aryl groups. Particularly preferredsubstituents at the 7-position are OMe and OCH₂Ph.

R² is preferably an optionally substituted C₅₋₇ aryl group, and mostpreferably an optionally substituted phenyl group.

In some embodiments, R² is a C₉₋₁₂ aryl group, for example napthy-1-ylor napth-2-yl. Further examples of C₉₋₁₂ aryl groups include quinolinyl,for example, quinolin-2-yl, quinolin-3-yl and quinolin-6-yl.

In other embodiments, R² is a C₅₋₇ heteroaryl group, for examplefuranyl, thiophenyl and pyridyl. Of these thiophenyl is preferred, forexample, thiophen-2-yl and thiophen-3-yl.

The C₅₋₂₀ aryl group may bear any substituent group. It preferably bearsfrom 1 to 3 substituent groups, with 1 and 2 being more preferred, andsingly substituted groups being most preferred.

Preferred C₅₋₂₀ aryl substituents, particularly for phenyl, include:halo (e.g. F, Cl, Br); C₁₋₇ alkoxy (e.g. methoxy, ethoxy); C₁₋₇ alkyl(e.g. methyl, trifluoromethyl, ethyl, propyl, t-butyl); bis-oxy-alkylene(e.g. bis-oxy-methylene, —O—CH₂—O—).

Particularly preferred substituted C₅₋₂₀ aryl groups include, but arenot limited to, 4-methyl-phenyl, 4-methoxy-phenyl, 3-methoxyphenyl,4-fluoro-phenyl, 3,4-bisoxymethylene-phenyl, 4-triflouoromethylphenyl,4-methylthiophenyl, 4-cyanophenyl and 4-phenoxyphenyl.

Particularly preferred unsubstituted C₅₋₂₀ aryl groups include, but arenot limited to thiophen-2-yl, napth-2-yl, quinolin-3-yl andquinolin-6-yl.

If R is optionally substituted C₁₋₁₂ alkyl, it is preferred that it isoptionally substituted C₁₋₇ alkyl.

It is preferred that both PBD monomers are identically substituted.

It is preferred that M and M′ are monovalent pharmaceutically acceptablecations, and are more preferably Na⁺.

z is preferably 3.

Preferred compounds include:

More preferred compounds include:

A most preferred compound is:

The above preferred compounds may also have a C₅ alkylene linking chain.

EXAMPLES Example 1 (a)(2S,4R)—N-(Benzyloxycarbonyl)-2-t-butyldimethylsilyloxymethyl-4-hydroxypyrrolidine(1)

Compound 1 is formed in high yield in a four step process known in theart starting from trans-4-hydroxy-L-proline (S. J. Gregson et al., J.Med. Chem., 2004, 1161-1174).

(b)(2S,4R)—N-(Benzyloxycarbonyl-2-t-butyldimethylsilyloxymethyl-4-oxyacetylpyrrolidine(2)

Pyridine (18.3 g, 18.7 mL, 232 mmol, 1.1 eq), acetic anhydride (23.6 g,21.8 mL, 232 mmol, 1.1 eq) and DMAP (5.14 g, 42.1 mmol, 0.2 eq) wereadded to a stirred solution of 1 (76.9 g, 211 mmol) in dry THF (1 L).The reaction mixture was stirred for 16 hours after which time TLC (95:5v/v CHCl₃/MeOH) showed the complete consumption of starting material.Excess solvent was removed by rotary evaporation and the residue wasdissolved in EtOAc (1 L), washed with 1N HCl (2×1 L), H₂O (1 L), brine(1 L) and dried (MgSO₄). Filtration and evaporation of the solventafforded acetate 2 as a colourless oil (80.7 g, 94%): ¹H NMR (400 MHz,CDCl₃) (rotamers) δ7.36-7.12 (m, 5H), 5.30-5.10 (m, 3H), 4.09-3.97 (m,2H), 3.74-3.55 (m, 3H), 2.36-2.29 (m, 1H), 2.11-2.06 (m, 1H), 2.02 (s,3H), 0.87 (s, 6H), 0.86 (s, 3H), 0.03 and 0.00 (s×2, 6H); MS (ES), m/z(relative intensity) 430 ([M+Na]⁺, 95), 408 ([M+H]⁺, 100).

(c) (2S,4R)-2-t-Butyldimethylsilyloxymethyl-4-oxyacetylpyrrolidine (3)

A slurry of silyl ether 2 (1.95 g, 4.80 mmol) and 10% Pd/C (0.17 g) inabsolute ethanol (10 mL) was subjected to Parr hydrogenation at 45 Psifor 16 h after which time TLC (95:5 v/v CHCl₃/MeOH) showed the completeconsumption of starting material. The reaction mixture was filteredthrough celite to remove the Pd/C, and the filter pad was washedrepeatedly with ethanol. Excess solvent was removed by rotaryevaporation under reduced pressure to afford the amine 3 as a paleorange waxy oil (1.28 g, 98%): IR (CHCl₃) 3315, 2930, 2858, 1739, 1652,1472, 1435, 1375, 1251, 1088, 838, 779, 667 cm⁻¹.

(d)1,1′-[[(Propane-1,3-diyl)dioxy]bis[(2S,4R)-(5-methoxy-2-nitro-1,4-phenylene)carbonyl]]bis[2-(tert-butyldimethylsilyloxymethyl)-4-oxyacetylpyrrolidine](5)

A catalytic amount of DMF (2 drops) was added to a stirred solution ofthe nitro-acid 4 (8.12 g, 17.4 mmol)¹ and oxalyl chloride (3.80 mL, 5.52g, 43.5 mmol, 2.5 eq) in dry THF (250 mL). The initial precipitatedissolved gradually and the reaction mixture was allowed to stir for 16h at room temperature. The resulting acid chloride solution was addeddropwise to a stirred mixture of the amine 3 (11.9 g, 43.5 mmol, 2.5eq), TEA (9.71 mL, 7.05 g, 69.7 mmol, 4.0 eq) and H₂O (2.26 mL) in THF(100 mL) at 0° C. (ice/acetone) under a nitrogen atmosphere. Thereaction mixture was allowed to warm to room temperature and stirred fora further 2.5 h. Excess THF was removed by rotary evaporation and theresulting residue was partitioned between H₂O (400 mL) and EtOAc (400mL). The layers were allowed to separate and the aqueous layer wasextracted with EtOAc (3×200 mL). The combined organic layers were thenwashed with saturated NH₄Cl (200 mL), saturated NaHCO₃ (200 mL), brine(200 mL) and dried (MgSO₄). Filtration and evaporation of the solventgave the crude product as a dark oil. Purification by flashchromatography (99.7:0.3 v/v CHCl₃/MeOH) isolated the pure amide 5 as alight yellow glass (13.3 g, 78%): ¹H NMR (400 MHz, CDCl₃) δ 7.60 (s,2H), 6.60 (s, 2H), 5.06 (br s, 2H), 4.44 (br s, 2H), 4.25-4.20 (m, 4H),4.10-4.08 (m, 2H), 3.80 (s, 6H), 3.64-3.62 (m, 2H), 3.36-3.32 (m, 2H),3.11-3.08 (m, 2H), 2.36-2.26 (m, 4H), 2.13-2.08 (m, 2H), 1.92 (s, 6H),0.80 (s, 18H), 0.00 (s×2, 12H); ¹³C NMR (100.6 MHz, CDCl₃) δ 171.0,166.3, 154.5, 148.2, 137.4, 128.0, 127.2, 109.2, 108.5, 72.9, 65.6,62.6, 57.4, 56.5, 54.8, 33.0, 28.6, 25.8, 21.0, 18.1; MS (ES), m/z(relative intensity) 1000 ([M+Na]⁺, 39), 978 ([M+H]⁺, 63), 977 (M⁺,100), 812 (13).

(e)1,1′-[[(Propane-1,3-diyl)dioxy]bis[(2S,4R)-(5-methoxy-2-amino-1,4-phenylene)carbonyl]]bis[2-(tert-butyldimethylsilyloxymethyl)-4-oxyacetylpyrrolidine](6)

Sodium dithionite (16.59 g, 95.27 mmol, 5 eq) was added to a stirredsolution of amide 5 (18.6 g, 19.1 mmol) in H₂O (200 mL) and THF (400mL). The reaction mixture was allowed to stir for 36 h after which timeexcess THF was removed by rotary evaporation and the resulting residuewas extracted with EtOAc (3×250 mL). The combined organic layers werethen washed with H₂O (300 mL), brine (300 mL) and dried (MgSO₄).Filtration and evaporation of the solvent yielded the crude productwhich was purified by flash:chromatography (80:20 v/v hexane/EtOAc thengradient to neat EtOAc) to afford the product 6 as a yellow foam (9.53g, 55%): ¹H NMR (400 MHz, CDCl₃) (rotamers) δ 6.70 and 6.67 (s×2, 2H),6.25 and 6.23 (s×2, 2H), 5.20 (br s, 2H), 4.49 (br s, 4H), 4.16-4.05 (m,6H), 3.70 (s, 6H), 3.68-3.57 (m, 4H), 2.36-2.27 (m, 4H), 2.12-2.04 (m,2H), 1.96 (s, 6H), 0.85 (s, 18H), 0.01 and 0.00 (s×2, 12H); ¹³C NMR(100.6 MHz, CDCl₃) δ 170.6, 170.0, 141.1, 116.3, 113.1, 102.3, 102.1,102.0, 66.2, 65.3, 65.2, 57.0, 28.9, 18.2; MS (ES), m/z (relativeintensity) 946 (M⁺+29, 43), 933 ([M+16]⁺, 61), 932 ([M+15]⁺, 100), 918([M+H]⁺, 72).

(f)1,1′-[[(Propane-1,3-diyl)dioxy]bis[(2S,4R)-[5-methoxy-1,4-phenylene-2-(2,2,2-trichloroethoxycarbonylamino)]carbonyl]]bis[2-(tert-butyldimethylsilyloxymethyl)-4-oxyacetylpyrrolidine](7)

A solution of 2,2,2-trichloroethyl chloroformate (3.58 mL, 5.50 g, 26.0mmol, 2.2 eq) in dry DCM (60 mL) was added dropwise to a solution ofanhydrous pyridine (3.82 mL, 3.80 g, 47.2 mmol, 4.0 eq) and bis-aniline6 (10.8 g, 11.8 mmol) in dry DCM (150 mL) at −10° C. (liq.N₂/ethanediol). After 16 h at room temperature, the reaction mixture waswashed with saturated NH₄Cl (2×150 mL), saturated CuSO₄ (150 mL), H₂O(150 mL), brine (150 mL) and dried (MgSO₄). Filtration and evaporationof the solvent yielded a yellow viscous oil which was purified by flashchromatography (70:30 v/v hexane/EtOAc) to afford the product 7 as awhite glass (13.8 g, 92%): ¹H NMR (400 MHz, CDCl₃) δ 9.42 (br s, 1H),7.83 (s, 2H), 6.76 and 6.74 (s×2, 2H), 5.21 (br s, 2H), 4.79 and 4.73(d×2, 4H, J=12.0 Hz), 4.56 (br s, 2H), 4.26-4.23 (m, 4H), 4.09-4.04 (m,2H), 3.74 (s, 6H), 3.72-3.68 (m, 2H), 3.60 (br s, 4H), 2.40-2.32 (m,4H), 2.23-2.08 (m, 2H), 1.95 (s, 6H), 0.85 (s, 18H), 0.01 and 0.00 (s×2,12H); ¹³C NMR (100.6 MHz, CDCl₃) δ 170.4, 169.2, 151.9, 151.5, 150.8,143.4, 132.6, 114.4, 111.7, 95.3, 74.4, 65.5, 65.4, 57.3, 56.4, 32.5,28.8, 25.8, 21.1, 18.1, 14.9; MS (ES), m/z (relative intensity) 1306([M+38]⁺, 92), 1304 ([M+36]⁺, 100), 1282 ([M+14]⁺, 97), 1280 ([M+12]⁺,55).

(g)1,1′-[[(Propane-1,3-diyl)dioxy]bis[(2S,4R)-[5-methoxy-1,4-phenylene-2-(2,2,2-trichloroethoxycarbonylamino)]carbonyl]]bis(2-hydroxymethyl-4-oxyacetylpyrrolidine)(8)

A mixture of glacial acetic acid (310 mL) and H₂O (100 mL) was added toa solution of 7 (13.8 g, 10.9 mmol) in THF (250 mL) and was stirred for16 h at room temperature. The reaction mixture was diluted with DCM (750mL) and neutralised with saturated NaHCO₃ (5 L). The aqueous layer wasextracted with DCM (3×500 mL) and the organic layers were combined,washed with brine (1 L) and dried (MgSO₄). TLC (60:40 v/v hexane/EtOAc)revealed the complete disappearance of the starting material. Filtrationand evaporation of the solvent afforded the crude product which waspurified by flash column chromatography (99.7:0.3 v/v CHCl₃/MeOH thengradient to 96:4 v/v CHCl₃/MeOH) to provide the product 8 as a whiteglass (11.6 g, >100%): ¹H NMR (500 MHz, CDCl₃) δ 8.92 (br s, 2H), 7.55(s, 1H), 6.71 (s, 1H), 5.18 (br s, 2H), 4.78 (d, 2H, J=12.0 Hz), 4.72(d, 2H, J=12.0 Hz), 4.50 (br s, 2H), 4.22-4.19 (m, 4H), 4.00 (br s, 2H),3.78 (s, 6H), 3.76-3.52 (m, 6H), 2.32-2.30 (m, 2H), 2.21-2.17 (m, 2H),2.09-2.04 (m, 2H) 1.94 (s, 6H); ¹³C NMR (125.8 MHz, CDCl₃) δ 170.4,152.2, 149.8, 145.0, 111.3, 106.5, 95.6, 74.4, 72.5, 65.4, 64.1, 58.7,56.5, 56.3, 33.6, 29.1, 21.1.

(h)1,1′-[[(Propane-1,3-diyl)dioxy]bis[(11S,11aS,2R)-10-(2,2,2-trichloroethoxycarbonyl)-11-hydroxy-7-methoxy-2-oxyacetyl-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5-one]](9)

TEMPO (0.69 g, 4.42 mmol, 0.4 eq) and BAIB (15.7 g, 48.7 mmol, 4.4 eq)were added to a stirred solution of diol 8 (11.5 g, 11.1 mmol) in DCM(150 mL). The reaction mixture was allowed to stir for 2 h and dilutedwith DCM (400 mL), washed with saturated NaHSO₃ (500 mL), saturatedNaHCO₃ (500 mL), brine (200 mL) and dried (MgSO₄). Filtration andevaporation of the solvent afforded the crude product which was purifiedby flash column chromatography (99.9:0.1 v/v CHCl₃/MeOH then gradient to99.7:0.3 v/v CHCl₃/MeOH) to provide the product 9 as a light yellowglass (4.43 g, 39%): ¹H NMR (400 MHz, CDCl₃) 7.28 (s, 2H, H6), 6.84 (s,2H, H9), 5.68 (d, 2H, J=9.1 Hz, H11), 5.37-5.35 (m, 2H, H2), 5.18 (d,2H, J=12.0 Hz, Troc CH₂), 4.32-4.21 (m, 6H, OCH₂CH₂CH₂O, Troc CH₂), 4.03(dd, 2H, J=13.2, 2.6 Hz, H3), 3.92 (s, 6H, OCH₃×2), 3.39-3.69 (m, 4H, H3and H11a), 2.39-2.35 (m, 6H, OCH₂CH₂CH₂O and H1), 2.03 (s, 6H,CH₃CO₂×2); ¹³C NMR (100.6 MHz, CDCl₃) δ 170.4 (CH₃CO₂), 167.4(C_(quat)), 154.3 (C_(quat)), 150.5 (C_(quat)), 149.1 (C_(quat)), 127.4(C_(quat)), 124.9 (C_(quat)), 114.1 (C9), 110.9 (C6), 95.0 (Troc CCl₃),87.5 (C11), 75.0 (Troc CH₂), 71.4 (C2), 65.5 (OCH₂CH₂CH₂O), 58.4 (C11a),56.1 (OCH₃), 51.1 (C3), 35.8 (C1), 29.1 (OCH₂CH₂CH₂O), 21.0 (CH₃CO₂); MS(ES), m/z (relative intensity) 1058 ([M+Na]⁺, 100).

(i)1,1′-[[(Propane-1,3-diyl)dioxy]bis[(11S,11aS,2R)-10-(2,2,2-trichloroethoxycarbonyl)-11-(tert-butyldimethylsilyloxy)-7-methoxy-2-oxyacetyl-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]](10)

TBSOTf (2.70 mL, 3.10 g, 11.7 mmol, 3.0 eq) was added to a stirredsolution of bis-alcohol 9 (4.05 g, 3.91 mmol) and 2,6-lutidine (1.82 mL,1.68 g, 15.6 mmol, 4.0 eq) in DCM (50 mL). The reaction mixture wasallowed to stir for 2.5 h and diluted with DCM (150 mL), washed withsaturated CuSO₄ (2×100 mL), saturated NaHCO₃ (100 mL), brine (200 mL)and dried (MgSO₄). Filtration and evaporation of the solvent affordedthe crude product which was purified by flash column chromatography(99.9:0.1 v/v CHCl₃/MeOH) to provide the product 10 as a white glass(5.05 g, >100%): ¹H NMR (400 MHz, CDCl₃) δ 7.05 (s, 2H, H6), 6.52 (s,2H, H9), 5.53 (d, 2H, J=9.0 Hz, H11), 5.14 (br s, 2H, H2), 4.99 (d, 2H,J=12.0 Hz, Troc CH₂), 4.06-3.87 (m, 8H, OCH₂CH₂CH₂O, Troc CH₂ and H11a),3.71 (s, 6H, OCH₃×2), 3.48-3.43 (m, 4H, H3), 2.21-2.11 (m, 4H,OCH₂CH₂CH₂O and H1), 2.03-1.96 (m, 2H, H1), 1.81 (s, 6H, CH₃CO₂×2), 0.63(s, 18H, TBS CH₃×6), 0.00 (s×2, 12H, TBS CH₃×4); ¹³C NMR (100.6 MHz,CDCl₃) δ 170.3 (CH₃CO₂), 167.9 (C_(quat)), 153.6 (C_(quat)), 150.4(C_(quat)), 149.2 (C_(quat)), 127.9 (C_(quat)), 125.5 (C_(quat)), 113.9(C9), 110.7 (C6), 95.2 (Troc CCl₃), 88.2 (C11), 74.7 (Troc CH₂), 71.7(C2), 65.0 (OCH₂CH₂CH₂O), 60.5 (C11a), 56.1 (OCH₃), 51.2 (C3), 36.2(C1), 28.8 (OCH₂CH₂CH₂O), 25.6 (TBS CH₃), 21.0 (CH₃CO₂), 17.8 (TBSC_(quat)), 14.2 and 14.1 (TBS CH₃); MS (ES), m/z (relative intensity)1285 ([M+21]⁺, 100), 1265 ([M+H]⁺, 75).

(j)1,1′-[[(Propane-1,3-diyl)dioxy]bis[(11S,11aS,2R)-10-(2,2,2-trichloroethoxycarbonyl)-11-(tert-butyldimethylsilyloxy)-7-methoxy-2-hydroxy-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]](11)

A solution of K₂CO₃ (93 mg, 0.67 mmol, 5.0 eq) in H₂O (2 mL) was addeddropwise to a stirred solution of acetate 10 (170 mg, 0.13 mmol) in MeOH(3 mL). The initial colorless solution eventually turned yellow and theformation of a white precipitate was observed. The reaction mixture wasallowed to stir for 16 h when TLC (95:5 v/v CHCl₃/MeOH) showed thecomplete consumption of the starting material. Excess solvent wasremoved by rotary evaporation and the mixture was carefully neutralizedwith 1N HCl to pH 7. The resulting mixture was extracted with EtOAc(3×25 mL) and the combined organic layers were then washed with brine(40 mL) and dried (MgSO₄). Filtration and removal of the solventafforded the product 11 as a white glass (151 mg, 95%): ¹H NMR (400 MHz,CDCl₃) δ 6.94 (s, 2H, H6), 6.52 (s, 2H, H9), 5.53 (d, 2H, J=9.0 Hz,H11), 5.00 (d, 2H, J=12.0 Hz, Troc CH₂), 4.36-4.35 (m, 2H, H2),4.06-3.82 (m, 8H, OCH₂CH₂CH₂O, Troc CH₂ and H3), 3.61 (s, 6H, OCH₃×2),3.54-3.48 (m, 2H, H11a), 3.39-3.34 (m, 2H, H3), 2.96 and 2.95 (br s×2,2H, OH×2), 2.21-2.20 (m, 2H, OCH₂CH₂CH₂O), 2.19-2.08 (m, 2H, H1),1.90-1.74 (m, 2H, H1), 0.64 (s, 18H, TBS CH₃×6), 0.00 (s, 12H, TBSCH₃×4); ¹³C NMR (100.6 MHz, CDCl₃) δ 168.5 (C_(quat)), 153.6 (C_(quat)),150.3 (C_(quat)), 149.1 (C_(quat)), 127.9 (C_(quat)), 125.4 (C_(quat)),113.9 (C9), 110.7 (C6), 95.2 (Troc CCl₃), 88.3 (C11), 74.7 (Troc CH₂),69.4 (C2), 65.0 (OCH₂CH₂CH₂O), 60.9 (C11a), 55.9 (OCH₃), 54.1 (C3), 38.8(C1), 28.9 (OCH₂CH₂CH₂O), 25.6 (TBS CH₃), 17.8 (TBS C_(quat)); MS (ES),m/z (relative intensity) 1196 ([M+16]⁺, 100), 1181 ([M+H]⁺, 82).

(k)1,1′-[[(Propane-1,3-diyl)dioxy]bis[(11S,11aS)-10-(2,2,2-trichloroethoxycarbonyl)-1′-(tert-butyldimethylsilyloxy)-7-methoxy-2-oxo-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]](12)

A solution of anhydrous DMSO (0.82 mL, 0.90 g, 11.5 mmol, 6.0 eq) in dryDCM (20 mL) was added dropwise to a stirred solution of oxalyl chloride(2.88 mL of a 2 M solution in DCM, 5.76 mmol, 3.0 eq) under a nitrogenatmosphere at −60° C. (liq N₂/CHCl₃). After stirring at −55° C. for 1.5h, a solution of the substrate 11 (2.26 g, 1.92 mmol) in dry DCM (30 mL)was added dropwise to the reaction mixture, which was then stirred for afurther 2 h at −45° C. A solution of TEA (10.8 mL, 7.82 g; 71.7 mmol,4.2 eq) in dry DCM (90 mL) was added dropwise to the mixture and stirredfor a further 30 min. The reaction mixture was left to warm to 0° C.,washed with cold 1 N HCl (2×50 mL), H₂O (50 mL), brine (50 mL) and dried(MgSO₄). Filtration and evaporation of the solvent in vacuo afforded thecrude product which was purified by flash column chromatography (70:30v/v hexane/EtOAc then gradient to 40:60 v/v hexane/EtOAc) to affordcarbinolamine 12 as a white glass (1.62 g, 72%): ¹H NMR (400 MHz, CDCl₃)δ 7.02 (s, 2H, H6), 6.54 (s, 2H, H9), 5.59 (d, 2H, J=9.2 Hz, H11), 4.98(d, 2H, J=12.0 Hz, Troc CH₂), 4.09-3.86 (m, 8H, OCH₂CH₂CH₂O, Troc CH₂and H3), 3.75-3.66 (m, 10H, OCH₃×2, H11a, and H3), 2.72 (dd, 2H, J=10.2,19.6 Hz, H1), 2.82 (dd, 2H, J=2.6, 19.6 Hz, H1), 2.22-2.19 (m, 2H,OCH₂CH₂CH₂O), 0.63 (s, 18H, TBS CH₃×6), 0.00 (s×2, 12H, TBS CH₃×4); ¹³CNMR (100.6 MHz, CDCl₃) δ 207.7 (C2), 168.0 (C_(quat)), 153.7 (C_(quat)),150.7 (C_(quat)), 149.4 (C_(quat)), 127.8 (C_(quat)), 124.6 (C_(quat)),114.0 (C9), 110.6 (C6), 95.1 (Troc CCl₃), 87.4 (C11), 74.8 (Troc CH₂),65.0 (OCH₂CH₂CH₂O), 58.9 (C11a), 56.1 (OCH₃), 53.0 (C3), 40.3 (C1), 28.8(OCH₂CH₂CH₂O), 25.6 (TBS CH₃), 17.8 (TBS C_(quat)); MS (ES), m/z(relative intensity) 1224 ([M+48]⁺, 100), 1210 ([M+34]⁺, 60), 1199([M+Na]⁺, 35), 1192 ([M+16]⁺, 40), 1176 (M⁺, 18).

(l)1,1′-[[(Propane-1,3-diyl)dioxy]bis[(11S,11aS)-10-(2,2,2-trichloroethoxycarbonyl)-11-(tert-butyldimethylsilyloxy)-7-methoxy-2-[[(trifluoromethyl)sulfonyl]oxy]-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]](13)

Anhydrous triflic anhydride (3.09 mL, 5.19 g, 18.4 mmol, 22 eq) takenfrom a freshly opened ampule was added rapidly in one portion to avigorously stirred solution of ketone 12 (0.98 g, 0.84 mmol) andanhydrous pyridine (1.49 mL, 1.46 g, 18.4 mmol, 22 eq) in dry DCM (50mL) at room temperature under a nitrogen atmosphere. The initialprecipitate dissolved gradually and the solution eventually turned adark red colour. The reaction mixture was allowed to stir for 4.5 h whenTLC (80:20 v/v EtOAc/hexane) revealed the complete consumption of thestarting material. The mixture was poured into cold saturated NaHCO₃ (60mL) and extracted with DCM (3×80 mL). The combined organic layers werethen washed with saturated CuSO₄ (2×125 mL), brine (125 mL) and dried(MgSO₄). Filtration and evaporation of the solvent afforded the crudeproduct which was purified by flash column chromatography (80:20 v/vhexane/EtOAc) to afford triflate 13 as a light yellow glass (0.74 mg,61%): [α]_(D) ²⁵=+46.0° (c=0.33, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 7.23(s, 2H, H6), 7.19 (s, 2H, H3), 6.77 (s, 2H, H9), 5.94 (d, 2H, J=8.9 Hz,H11), 5.23 (d, 2H, J=12.0 Hz, Troc CH₂), 4.31-4.28 (m, 2H, OCH₂CH₂CH₂O),4.18 (d, 2H, J=12.2 Hz, Troc CH₂), 4.15-4.13 (m, 2H, OCH₂CH₂CH₂O),3.95-3.91 (m, 8H, OCH₃×2, H11a), 3.35 (dd, 2H, J=11.0, 16.6 Hz, H1),2.84 (d, 2H, J=16.6 Hz, H1), 2.46-2.44 (m, 2H, OCH₂CH₂CH₂O), 0.89 (s,18H, TBS CH₃×6), 0.29 and 0.26 (s×2, 12H, TBS CH₃×4); ¹³C NMR (100.6MHz, CDCl₃) δ 164.9 (C_(quat)), 153.6 (C_(quat)), 151.0 (C_(quat)),149.5 (C_(quat)), 136.0 (C_(quat)), 127.7 (C_(quat)), 123.9 (C_(quat)),121.0 (C3), 114.0 (C9), 110.9 (C6), 95.1 (Troc CCl₃), 86.3 (C11), 74.8(Troc CH₂), 65.0 (OCH₂CH₂CH₂O), 60.6 (C11a), 56.2 (OCH₃), 34.4 (C1),28.8 (OCH₂CH₂CH₂O), 25.6 (TBS CH₃), 17.8 (TBS C_(quat)); IR (CHCl₃)3020, 2957, 2860, 1725, 1674, 1651, 1604, 1516, 1466, 1454, 1431, 1409,1329, 1312, 1274, 1216, 1138, 1113, 1083, 1042, 1006, 900, 840, 757,668, 646, 610 cm⁻¹; MS (ES), m/z (relative intensity) 1461 ([M+21]⁺,100), 1440 (M⁺, 55).

(m)1,1′-[[(Propane-1,3-diyl)dioxy]bis[(11S,11aS)-10-(2,2,2-trichloroethoxycarbonyl)-11-(tert-butyldimethylsilyloxy)-7-methoxy-2-(p-methoxybenzene)-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]](14)

A solution of TEA (0.20 mL, 148 mg, 1.46 mmol, 6.0 eq) in H₂O (1.5 mL)and EtOH (10 mL) was added to a solution of triflate 13 (350 mg, 0.24mmol) in toluene (10 mL) at room temperature. To this mixture4-methoxybenzeneboronic acid (96 mg, 0.63 mmol, 2.6 eq) and Pd(PPh₃)₄(11 mg, 9 μmol, 0.04 eq) were added. The reaction mixture was allowed tostir for 15 min when TLC (80:20 v/v EtOAc/hexane) revealed the completeconsumption of the starting material. Excess solvent was removed and theresidue was dissolved in EtOAc (25 mL), washed with H₂O (15 mL), brine(15 mL) and dried (MgSO₄). Filtration and evaporation of solventafforded the crude product which was purified by flash columnchromatography (80:20 v/v hexane/EtOAc then gradient to 50:50 v/vhexane/EtOAc) to afford 14 as a yellow glass (286 mg, 87%): ¹H NMR (400MHz, CDCl₃) δ 7.38 (s, 2H, H3), 7.32-7.28 (m, 6H, H6 and H13), 6.92 (d,4H, J=8.7 Hz, H14), 6.81 (s, 2H, H9), 5.93 (d, 2H, J=8.8 Hz, H11), 5.24(d, 2H, J=12.0 Hz, Troc CH₂), 4.34-4.29 (m, 2H, OCH₂CH₂CH₂O), 4.20-4.11(m, 4H, Troc CH₂ and OCH₂CH₂CH₂O), 4.00-3.96 (m, 8H, H11a and OCH₃×2),3.84 (s, 6H, OCH₃×2), 3.36 (dd, 2H, J=10.8, 16.6 Hz, H1), 2.85 (d, 2H,J=16.5 Hz, H1), 2.48-2.45 (m, 2H, OCH₂CH₂CH₂O), 0.93 (s, 18H, TBSCH₃×6), 0.30 and 0.27 (s×2, 12H, TBS CH₃×4); ¹³C NMR (100.6 MHz, CDCl₃)δ 162.5 (C_(quat)), 161.3 (C_(quat)), 159.2 (C_(quat)), 151.1(C_(quat)), 148.1 (C_(quat)), 140.3 (C_(quat)), 126.2 (C13), 126.0(C_(quat)), 123.2 (C_(quat)), 121.9 (C3), 119.3 (C_(quat)), 114.3 (C6),111.9 (C14), 111.2 (C9), 95.2 (Troc CCl₃), 87.3 (C11), 74.8 (Troc CH₂),65.0 (OCH₂CH₂CH₂O), 61.5 (C11a), 56.1 and 55.3 (OCH₃), 35.3 (C1), 28.8(OCH₂CH₂CH₂O), 25.7 (TBS CH₃), 17.9 (TBS C_(quat)); MS (ES), m/z(relative intensity) 1357 (M⁺, 63), 1114 (48), 955 (59), 919 (78).

(n)1,1′-[[(Propane-1,3-diyl)dioxy]bis[(11aS)-7-methoxy-2-(p-methoxybenzene)-1,11a-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]](ZC-207)

10% Cd/Pd couple (461 mg, 3.73 mmol, 20 eq) was added to a rapidlystirring mixture of 14 (253 mg, 0.19 mmol), THF (5 mL) and 1 N NH₄OAc (5mL). The reaction mixture was allowed to stir for 1.5 h when TLC showedthe complete consumption of the starting material. The solids werefiltered and rinsed with H₂O and DCM. The aqueous layer was extractedwith DCM (3×30 mL) and the organic extracts were combined, washed withbrine (50 mL) and dried (MgSO₄). Filtration and evaporation of solventafforded the crude product which was purified by flash columnchromatography (99.9:0.1 v/v CHCl₃/MeOH then gradient to 95:5 v/vCHCl₃/MeOH) to afford ZC-207 as a yellow glass (132 mg, 96%): [α]_(D)²⁰=+880.0° (c=0.22, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 7.79 (d, 2H, J=3.9Hz, H11), 7.44 (s, 2H, H6), 7.30 (s, 2H, H3), 7.24 (d, 4H, J=8.7 Hz,H13), 6.81 (d, 4H, J=8.7 Hz, H14), 6.79 (s, 2H, H9), 4.30-4.18 (m, 6H,OCH₂CH₂CH₂O and H11a), 3.86 (s, 6H, OCH₃×2), 3.74 (s, 6H, OCH₃×2), 3.48(dd, 2H, J=11.8, 16.2 Hz, H1), 2.85 (d, 2H, J=16.2 Hz, H1), 2.38-2.32(m, 2H, OCH₂CH₂CH₂O); ¹³C NMR (62.9 MHz, CDCl₃) δ 162.5 (C11), 161.3(C_(quat)), 159.2 (C_(quat)), 151.1 (C_(quat)), 148.1 (C_(quat)), 140.3(C_(quat)), 126.2 (C13), 126.0 (C_(quat)), 123.2 (C_(quat)), 121.9 (C3),114.3 (C14), 111.9 (C9), 111.2 (C6), 65.4 (OCH₂CH₂CH₂O), 56.2 and 55.3(OCH₃), 53.8 (C11a), 35.6 (C1), 28.9 (OCH₂CH₂CH₂O); MS (ES), m/z(relative intensity) 741 (M⁺, 43), 660 (71).

(o)1,1′[[(Propane-1,5-diyl)dioxy]bis[(11aS)-11-sulpho-7-methoxy-2-(4-methoxyphenyl)-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]]sodium salt (ZC-423)

A solution of sodium bisulphite (6.74 mg, 65 μmol) in water (1.5 mL) wasadded to a stirred solution of ZC-207 (24.01 mg, 32 μmol) indichloromethane (1.5 mL). The reaction mixture was allowed to stirvigorously for 2 h, after which time the organic and aqueous layers wereseparated. TLC analysis (eluent-EtOAc) of the aqueous phase revealedabsence of ZC-207 and presence of baseline material with strong uvabsorption. The aqueous layer was lyophilised to provide the bisulphiteadduct ZC-423 as a white solid (17 mg, 55%): ¹H NMR (400 MHz, d₆-DMSO) δ7.42 (s, 2H, H-3), 7.38 (d, 4H, J=8.72 Hz, 2′-H), 7.05 (s, 2H, H-6),6.92 (d, 4H, J=8.92 Hz, 3′-H), 6.52 (s, 2H, H-9), 5.27 (s, 2H, NH),4.35-4.25 (m, 2H, H11a), 4.15-4.05 (m, 4H, OCH₂CH₂CH₂O), 3.95 (d, 2H,J=10.4 Hz, H11), 3.77 (s, 6H, OMe), 3.72 (s, 6H, OMe), 3.55-3.45 (m, 2H,H1), 3.30-3.15 (m, 2H, H1), 2.25-2.15 (m, 2H, OCH₂CH₂CH₂O).

Example 2 (a)1,1′-[[(Propane-1,3-diyl)dioxy]bis[(11S,11aS)-10-(2,2,2-trichloroethoxycarbonyl)-11-(tert-butyldimethylsilyloxy)-7-methoxy-2-(naphthalen-2-yl)-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]](17)

A solution of TEA (0.15 mL, 1.05 mmol, 6.0 eq) in H₂O (1 mL) and EtOH(10 mL) was added to a solution of 13 (251 mg, 0.17 mmol) in toluene (6mL) at room temperature. To this mixture 2-naphthaleneboronic acid (77.9mg, 0.45 mmol, 2.6 eq) and Pd(PPh₃)₄ (8.0 mg, 7 mmol, 0.04 eq) wereadded. The reaction mixture was heated at 100° C. under microwaveirradiation for 20 minutes when TLC (80:20 v/v EtOAc/hexane) revealedcomplete consumption of the starting material. Excess solvent wasremoved and the residue was dissolved in EtOAc (20 mL), washed with H₂O(15 mL), brine (15 mL) and dried (MgSO₄). Filtration and evaporation ofsolvent afforded the crude product which was purified by flash columnchromatography (80:20 v/v hexane/EtOAc then gradient to 50:50 v/vhexane/EtOAc) to afford 17 as a yellow glass (191 mg, 81%): ¹H NMR (400MHz, CDCl₃) δ7.32-7.29 (m, 6H, H_(arom)), 7.61-7.58 (m, 6H, H_(arom)),7.52-7.42 (m, 4H, H3 and H_(arom)), 7.30 (s, 2H, H6), 6.81 (s, 2H, H9),5.98 (d, 2H, J=8.8 Hz, H11), 5.24 (d, 2H, J=12.0 Hz, Troc CH₂),4.35-4.30 (m, 2H, OCH₂CH₂CH₂O), 4.20-4.13 (m, 4H, Troc CH₂ andOCH₂CH₂CH₂O), 4.08-4.00 (m, 2H, H11a), 3.97 (s, 6H, OCH₃×2), 3.50 (dd,2H, J=10.8, 16.6 Hz, H1), 2.98 (d, 2H, J=16.5 Hz, H1), 2.50-2.48 (m, 2H,OCH₂CH₂CH₂O), 0.94 (s, 18H, TBS CH₃×6), 0.30 and 0.28 (s×2, 12H, TBSCH₃×4).

(b)1,1′-[[(Propane-1,3-diyl)dioxy]bis[(11aS)-7-methoxy-2-(naphthalen-2-yl)-1,11a-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]](18)

10% Cd/Pb couple (455 mg, 3.71 mmol, 26 eq) was added to a rapidlystirring mixture of 17 (190 mg, 0.14 mmol), THF (3.2 mL) and 1 N NH₄OAc(3.2 mL). The reaction mixture was allowed to stir for 6.5 hours whenTLC showed the incomplete consumption of the starting material andformation of side products. The solids were filtered and rinsed with H₂Oand MeOH. Excess solvent was removed and the residue was diluted withH₂O (25 mL) and DCM (25 mL). The aqueous layer was extracted with DCM(3×25 mL) and the organic extracts were combined, washed with brine (50mL) and dried (MgSO₄). Filtration and evaporation of solvent affordedthe crude product which was purified by flash column chromatography(99:1 v/v CHCl₃/MeOH then gradient to 98:2 v/v CHCl₃/MeOH) to afford 18as a yellow glass (38 mg, 36%): ¹H NMR (400 MHz, CDCl₃) δ 7.85 (d, 2H,J=3.9 Hz, H11), 7.77-7.65 (m, 6H, H_(arom)), 7.59-7.50 (m, 6H, H3 andH_(arom)), 7.47 (s, 2H, H6), 7.44-7.33 (m, 4H, H_(arom)), 6.82 (s, 2H,H9), 4.42-4.32 (m, 2H, H11a), 4.31-4.14 (m, 4H, OCH₂CH₂CH₂O), 3.89 (s,6H, OCH₃×2), 3.69-3.56 (m, 2H, H1), 3.50-3.37 (m, 2H, H1), 2.45-2.29 (m,2H, OCH₂CH₂CH₂O).

(c)1,1′-[[(Propane-1,3-diyl)dioxy]bis[(11aS)-11-sulpho-7-methoxy-2-(naphthalen-2-yl)-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]]sodium salt (19)

A solution of sodium bisulphite (6.67 mg, 0.064 mmol, 2.0 equiv.) in H₂O(1.5 mL) was added to a solution of compound 18 (25 mg, 0.032 mmol) indry DCM (1.5 mL). The reaction mixture was allowed to stir for 5 hours.The reaction mixture was diluted with DCM (5 mL) and H₂O (5 mL). Theaqueous layer was separated (without shaking the separating funnel) anddried under the freeze dryer to afford 19 as a lightweight white solid(7 mg, 22%): ¹H NMR (400 MHz, DMSO) δ 7.78-7.72 (m, 8H, H_(arom)), 7.68(s, 2H, H3), 7.59-7.27 (m, 6H, H_(arom)), 6.92 (s, 2H, H6), 6.41 (s, 2H,H9), 5.21 (d, 2H, J=5.2 Hz, NH), 4.38-4.31 (m, 2H, H11a), 4.12-3.96 (m,4H, OCH₂CH₂CH₂O), 3.87 (d, 2H, J=10.4 Hz, H11), 3.70 (s, 3H, OCH₃), 3.61(s, 3H, OCH₃), 3.40-3.12 (m, 4H, H1), 2.28-2.22 (m, 2H, OCH₂CH₂CH₂O).

Example 3 (a)1,1′-[[(Propane-1,3-diyl)dioxy]bis[(11S,11aS)-10-(2,2,2-trichloroethoxycarbonyl)-11-(tert-butyldimethylsilyloxy)-7-methoxy-2-(thiophen-2-yl)-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]](20)

A solution of TEA (0.11 mL, 0.82 mmol, 6.0 eq) in H₂O (0.8 mL) and EtOH(5 mL) was added to a solution of 13 (197 mg, 0.14 mmol) in toluene (5mL). To this mixture thiophene-2-boronic acid (45.5 mg, 0.36 mmol, 2.6eq) and Pd(PPh₃)₄ (6.3 mg, 5 μmol, 0.04 eq) were added. The reactionmixture was allowed to stir at room temperature for 3 hours when TLC(80:20 v/v EtOAc/hexane) revealed complete consumption of the startingmaterial. Excess solvent was removed and the residue was dissolved inEtOAc (20 mL), washed with H₂O (15 mL), brine (15 mL) and dried (MgSO₄).Filtration and evaporation of solvent afforded the crude product whichwas purified by flash column chromatography (80:20 v/v hexane/EtOAc) toafford 20 as a yellow glass (168 mg, 94%): ¹H NMR (400 MHz, CDCl₃) δ7.32 (s, 2H, H3), 7.26 (s, 2H, H6), 7.24-7.19 (m, 2H, H_(arom)),7.04-6.79 (m, 2H, H_(arom)), 6.94-6.88 (m, 2H, H_(arom)), 6.78 (s, 2H,H9), 5.92 (d, 2H, J=8.9 Hz, H11), 5.24 (d, 2H, J=12.0 Hz, Troc CH₂),4.36-4.25 (m, 2H, OCH₂CH₂CH₂O), 4.19-4.08 (m, 4H, Troc CH₂ andOCH₂CH₂CH₂O), 4.02-3.87 (m, 8H, H11a and OCH₃×2), 3.37 (dd, 2H, J=10.8,16.6 Hz, H1), 2.85 (d, 2H, J=16.5 Hz, H1), 2.52-2.37 (m, 2H,OCH₂CH₂CH₂O), 0.91 (s, 18H, TBS CH₃×6), 0.28 and 0.25 (s×2, 12H, TBSCH₃×4).

(b)1,1′-[[(Propane-1,3-diyl)dioxy]bis[(11aS)-7-methoxy-2-(thiophen-2-yl)-1,11a-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]](21)

10% Cd/Pb couple (288 mg, 2.35 mmol, 20 eq) was added to a rapidlystirring mixture of 20 (154 mg, 0.12 mmol), THF (3 mL) and 1 N NH₄OAc (3mL). The reaction mixture was allowed to stir for 3 hours when TLCshowed complete consumption of the starting material. The solids werefiltered and rinsed with H₂O and DCM. The aqueous layer was extractedwith DCM (3×15 mL) and the organic extracts were combined, washed withbrine (40 mL) and dried (MgSO₄). Filtration and evaporation of solventafforded the crude product which was purified by flash columnchromatography (99:1 v/v CHCl₃/MeOH then gradient to 98:2 v/vCHCl₃/MeOH) to afford 21 as a yellow glass (59 mg, 72%): ¹H NMR (400MHz, CDCl₃) δ 7.90 (d, 2H, J=4.0 Hz, H11), 7.51 (s, 2H, H6), 7.36 (s,2H, H3), 7.22 (d, 2H, J=5.2 Hz, H_(arom)), 7.02 (dd, 2H, J=3.6, 5.0 Hz,H_(arom)), 6.98 (d, 2H, J=3.4 Hz, H_(arom)), 6.88 (s, 2H, H9), 4.43-4.23(m, 6H, H11a and OCH₂CH₂CH₂O), 3.94 (s, 6H, OCH₃×2), 3.59 (ddd, 2H,J=2.0, 11.5, 16.0 Hz, H1), 2.85 (ddd, 2H, J=1.5, 5.2, 16.0 Hz, H1),2.49-2.40 (m, 2H, OCH₂CH₂CH₂O).

(c)1,1′-[[(Propane-1,3-diyl)dioxy]bis[(11aS)-11-sulpho-7-methoxy-2-(thiophen-2-yl)-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]] sodiumsalt (22)

A solution of sodium bisulphite (16.5 mg, 0.159 mmol, 2.0 equiv.) in H₂O(3.5 mL) was added to a solution of compound 21 (55.0 mg, 0.079 mmol) inDCM (3.5 mL). The reaction mixture was allowed to stir for 4 hours. TLC(EtOAc) revealed complete consumption of the starting material and aproduct spot at the base line. The reaction mixture was diluted with DCMand H₂O. The aqueous layer was separated (without shaking the separatingfunnel) and dried under the freeze dryer to afford 22 as a lightweightyellow solid (53.9 mg, 75%): ¹H NMR (400 MHz, DMSO) δ 7.42 (dd, 2H,J=1.5, 4.7 Hz, H_(arom)), 7.33-7.30 (m, 6H, H_(arom)), 6.53 (s, 2H, H9),5.30 (d, 2H, J=3.8 Hz, NH), 4.35-4.29 (m, 2H, H11a), 4.12-4.06 (m, 4H,OCH₂CH₂CH₂O), 3.95 (d, 2H, J=10.3 Hz, H11), 3.72 (s, 6H, OCH₃×2),3.53-3.19 (m, 4H, H1), 2.23-2.21 (m, 2H, OCH₂CH₂CH₂O).

Example 4 (a)1,1′-[[(Propane-1,3-diyl)dioxy]bis[(11S,11aS)-10-(2,2,2-trichloroethoxycarbonyl)-11-(tert-butyldimethylsilyloxy)-7-methoxy-2-(quinolin-6-yl)-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]](23)

A solution of TEA (0.12 mL, 0.84 mmol, 6.0 eq) in H₂O (0.8 mL) and EtOH(5 mL) was added to a solution of triflate 13 (202 mg, 0.14 mmol) intoluene (5 mL) at room temperature. To this mixture6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) quinoline (93.0 mg, 0.36mmol, 2.6 eq) and Pd(PPh₃)₄ (6.5 mg, 6 μmol, 0.04 eq) were added. Thereaction mixture was heated at 100° C. under microwave irradiation for15 minutes when TLC (80:20 v/v EtOAc/hexane) revealed completeconsumption of the starting material. Excess solvent was removed and theresidue was dissolved in EtOAc (20 mL), washed with H₂O (15 mL), brine(15 mL) and dried (MgSO₄). Filtration and evaporation of solventafforded the crude product which was purified by flash columnchromatography (80:20 v/v hexane/EtOAc then gradient to 50:50 v/vhexane/EtOAc) to afford 23 as a yellow glass (126 mg, 64%): ¹H NMR (400MHz, CDCl₃) δ 8.88 (dd, 2H, J=1.6, 4.2 Hz, H_(arom)), 8.14 (d, 2H, J=7.6Hz, H_(arom)), 8.07 (d, 2H, J=9.0 Hz, H_(arom)), 7.85 (dd, 2H, J=1.8,8.9 Hz, H_(arom)), 7.66 (s, 2H, H3), 7.55 (d, 2H, J=1.8 Hz, H_(arom)),7.41 (dd, 2H, J=4.3, 8.3 Hz, H_(arom)), 7.31 (s, 2H, H6), 6.82 (s, 2H,H9), 5.98 (d, 2H, J=8.8 Hz, H11), 5.25 (d, 2H, J=12.1 Hz, Troc CH₂),4.34-4.31 (m, 2H, OCH₂CH₂CH₂O), 4.21-4.15 (m, 4H, Troc CH₂ andOCH₂CH₂CH₂O), 4.09-4.03 (m, 2H, H11a), 3.97 (s, 6H, OCH₃×2), 3.48 (ddd,2H, J=1.6, 10.4, 16.1 Hz, H1), 2.98 (dd, 2H, J=2.4, 16.1 Hz, H1),2.50-2.47 (m, 2H, OCH₂CH₂CH₂O), 0.95 (s, 18H, TBS CH₃×6), 0.32 and 0.28(s×2, 12H, TBS CH₃×4).

(b)1,1′-[[(Propane-1,3-diyl)dioxy]bis[(11aS)-7-methoxy-2-(quinolin-6-yl)-1,11a-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]](24)

10% Cd/Pb couple (516 mg, 4.21 mmol, 25 eq) was added to a rapidlystirring mixture of 23 (234 mg, 0.17 mmol), THF (5 mL) and 1 N NH₄OAc (5mL). The reaction mixture was allowed to stir for 5 hours when TLCshowed complete consumption of the starting material and formation of asmall amount of side products. The solids were filtered and rinsed withH₂O and a large amount of MeOH. Excess solvent was removed and theresidue was diluted with H₂O (35 mL) and CHCl₃ (35 mL). The aqueouslayer was extracted with CHCl₃ (3×30 mL) and the organic extracts werecombined, washed with brine (100 mL) and dried (MgSO₄). Filtration andevaporation of solvent afforded the crude product which was purified byflash column chromatography (99.5:0.5 v/v CHCl₃/MeOH then gradient to96:4 v/v CHCl₃/MeOH) to afford 24 as a yellow glass (44 mg, 33%): ¹H NMR(400 MHz, CDCl₃) 8.85 (dd, 2H, J=1.6, 4.2 Hz, H_(arom)), 8.09 (d, 2H,J=7.6 Hz, H_(arom)), 8.03 (d, 2H, J=9.0 Hz, H_(arom)), 7.88 (d, 2H,J=3.9 Hz, H11), 7.82 (dd, 2H, J=1.8, 8.9 Hz, H_(arom)), 7.24 (s, 2H,H3), 7.59 (s, 2H, H_(arom)), 7.52 (s, 2H, H6), 7.48 (dd, 2H, J=4.3, 8.3Hz, H_(arom)), 6.88 (s, 2H, H9), 4.48-4.42 (m, 2H, H11a), 4.39-4.28 (m,4H, OCH₂CH₂CH₂O), 3.94 (s, 6H, OCH₃×2), 3.76-3.63 (m, 2H, H1), 3.09-2.98(m, 2H, H1), 2.46-2.43 (m, 2H, OCH₂CH₂CH₂O).

Example 5 (a)1,1′-[[(Propane-1,3-diyl)dioxy]bis[(11S,11aS)-10-(2,2,2-trichloroethoxycarbonyl)-11-(tert-butyldimethylsilyloxy)-7-methoxy-2-(3-methoxybenzene)-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]](25)

A solution of TEA (0.071 mL, 0.51 mmol, 6.0 eq) in H₂O (0.6 mL) and EtOH(3.5 mL) was added to a solution of triflate 13 (122 mg, 0.085 mmol) intoluene (3.5 mL) at room temperature. To this mixture3-methoxyphenolboronic acid (33.5 mg, 0.22 mmol, 2.6 eq) and Pd(PPh₃)₄(3.9 mg, 3.4 μmol, 0.04 eq) were added. The reaction mixture was heatedat 100° C. under microwave irradiation for 5 minutes when TLC (80:20 v/vEtOAc/hexane) revealed complete consumption of the starting material.Excess solvent was removed and the residue was dissolved in EtOAc (10mL), washed with H₂O (10 mL), brine (10 mL) and dried (MgSO₄).Filtration and evaporation of solvent afforded the crude product whichwas purified by flash column chromatography (80:20 v/v hexane/EtOAc thengradient to 60:40 v/v hexane/EtOAc) to afford 25 as a yellow glass (66mg, 57%): ¹H NMR (400 MHz, CDCl₃) 7.23 (s, 2H, H3), 7.05-7.00 (m, 4H, H6and H_(arom)), 6.70 (d, 2H, J=8.7 Hz, H_(arom)), 6.64 (s, 2H, H_(arom)),6.67-6.64 (m, 4H, H_(arom) and H9), 5.67 (d, 2H, J=8.8 Hz, H11), 4.98(d, 2H, J=12.0 Hz, Troc CH₂), 4.08-4.02 (m, 2H, OCH₂CH₂CH₂O), 3.92-3.84(m, 4H, Troc CH₂ and OCH₂CH₂CH₂O), 3.74-3.66 (m, 8H, H11a and OCH₃×2),3.68 (s, 6H, OCH₃×2), 3.10 (dd, 2H, J=10.8, 16.6 Hz, H1), 2.60 (d, 2H,J=16.5 Hz, H1), 2.22-2.19 (m, 2H, OCH₂CH₂CH₂O), 0.68 (s, 18H, TBSCH₃×6), 0.02 and 0.00 (s×2, 12H, TBS CH₃×4).

(b)1,1′-[[(Propane-1,3-diyl)dioxy]bis[(11aS)-7-methoxy-2-(3-methoxybenzene)-1,11a-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]](26)

10% Cd/Pb couple (269 mg, 2.20 mmol, 25 eq) was added to a rapidlystirring mixture of 25 (119 mg, 0.088 mmol), THF (3 mL) and 1 N NH₄OAc(3 mL). The reaction mixture was allowed to stir for 3.5 hours when TLCshowed complete consumption of the starting material. The solids werefiltered and rinsed with H₂O and MeOH. Excess solvent was removed andthe residue was diluted with H₂O (20 mL) and DCM (20 mL). The aqueouslayer was extracted with DCM (3×20 mL) and the organic extracts werecombined, washed with H₂O (50 mL) and brine (50 mL) and dried (MgSO₄).Filtration and evaporation of solvent afforded the crude product whichwas purified by flash column chromatography (99.9:0.1 v/v CHCl₃/MeOHthen gradient to 98:2 v/v CHCl₃/MeOH) to afford 26 as a yellow glass (35mg, 54%): NMR (400 MHz, CDCl₃) δ 7.79 (d, 2H, J=3.9 Hz, H11), 7.44 (s,2H, H3), 7.42 (s, 2H, H6), 7.21-7.16 (m, 2H, H_(arom)), 6.90 (d, 2H,J=8.7 Hz, H_(arom)), 6.84-6.83 (m, 2H, H_(arom)), 6.80 (s, 2H, H9),4.33-4.17 (m, 6H, OCH₂CH₂CH₂O and H11a), 3.86 (s, 6H, OCH₃×2), 3.75 (s,6H, OCH₃×2), 3.44 (dd, 2H, J=11.8, 16.2 Hz, H1), 3.30 (d, 2H, J=16.2 Hz,H1), 2.39-2.33 (m, 2H, OCH₂CH₂CH₂O).

(c)1,1′-[[(Propane-1,3-diyl)dioxy]bis[(11aS)-11-sulpho-7-methoxy-2-(3-methoxybenzene)-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]] sodiumsalt (27)

A solution of sodium bisulphite (7.6 mg, 0.073 mmol, 2.0 equiv.) in H₂O(1.7 mL) was added to a solution of compound 26 (27.0 mg, 0.036 mmol) inDCM (1.7 mL). The reaction mixture was allowed to stir for 2 hours. TLC(EtOAc) revealed complete consumption of the starting material and aproduct spot at the base line. The reaction mixture was diluted with DCM(5 mL) and H₂O (5 mL). The aqueous layer was separated and lyophilizedto afford 27 as a lightweight white solid (12.1 mg, 35%): ¹H NMR (400MHz, DMSO) δ 7.61 (s, 2H, H3), 7.27-7.23 (m, 2H, H_(arom)), 7.08-6.99(m, 6H, H_(arom) and H6), 6.81-6.78 (m, 2H, H_(arom)), 6.54 (s, 2H, H9),5.30 (s, 2H, NH), 4.37-4.30 (m, 2H, H11a), 4.17-4.08 (m, 4H,OCH₂CH₂CH₂O), 3.84 (d, 2H, J=10.4 Hz, H11), 3.78 (s, 6H, OCH₃×2), 3.62(s, 6H, OCH₃×2), 3.52-3.20 (m, 4H, H1), 2.26-2.20 (m, 2H, OCH₂CH₂CH₂O).

Example 6 (a)1,1′-[[(Propane-1,3-diyl)dioxy]bis[(11S,11aS)-10-(2,2,2-trichloroethoxycarbonyl)-11-(tert-butyldimethylsilyloxy)-7-methoxy-2-(3,4-methylenedioxyphenyl)-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]](28)

A solution of TEA (0.066 mL, 0.47 mmol, 6.0 eq) in H₂O (0.6 mL) and EtOH(3.5 mL) was added to a solution of triflate 13 (122 mg, 0.085 mmol) intoluene (3.5 mL) at room temperature. To this mixture3,4-methylenedioxyphenylboronic acid (34.2 mg, 0.21 mmol, 2.6 eq) andPd(PPh₃)₄ (3.6 mg, 3.2 μmol, 0.04 eq) were added. The reaction mixturewas heated at 100° C. under microwave irradiation for 5 minutes when TLC(80:20 v/v EtOAc/hexane) revealed complete consumption of the startingmaterial. Excess solvent was removed and the residue was dissolved inEtOAc (10 mL), washed with H₂O (10 mL), brine (10 mL) and dried (MgSO₄).Filtration and evaporation of solvent afforded the crude product whichwas purified by flash column chromatography (80:20 v/v hexane/EtOAc thengradient to 65:35 v/v hexane/EtOAc) to afford 28 as a yellow glass (84mg, 77%): ¹H NMR (400 MHz, CDCl₃) δ 7.34 (s, 2H, H6), 7.26 (s, 2H, H3),6.89 (s, 2H, H9), 6.78-6.76 (m, 4H, H_(arom)), 5.98 (s, 2H, OCH₂O), 5.89(d, 2H, J=8.8 Hz, H11), 5.23 (d, 2H, J=12.0 Hz, Troc CH₂), 4.32-4.27 (m,2H, OCH₂CH₂CH₂O), 4.18-4.09 (m, 4H, Troc CH₂ and OCH₂CH₂CH₂O), 3.97-3.91(m, 8H, H11a and OCH₃×2), 3.30 (dd, 2H, J=10.8, 16.6 Hz, H1), 2.76 (d,2H, J=16.6 Hz, H1), 2.46-2.44 (m, 2H, OCH₂CH₂CH₂O), 0.88 (s, 18H, TBSCH₃×6), 0.28 and 0.25 (s×2, 12H, TBS CH₃×4).

(b)1,1′-[[(Propane-1,3-diyl)dioxy]bis[(11aS)-7-methoxy-2-(3,4-methylenedioxyphenyl)-1,11a-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]](29)

10% Cd/Pb couple (307 mg, 2.51 mmol, 25 eq) was added to a rapidlystirring mixture of 28 (139 mg, 0.101 mmol), THF (3.5 mL) and 1 N NH₄OAc(3.5 mL). The reaction mixture was allowed to stir for 5 hours when TLCshowed complete consumption of the starting material and formation of asmall amount of side products. The solids were filtered and rinsed withH₂O and MeOH. Excess solvent was removed and the residue was dilutedwith H₂O (25 mL) and DCM (25 mL). The aqueous layer was extracted withDCM (3×40 mL) and the organic extracts were combined, washed with H₂O(80 mL) and brine (80 mL) and dried (MgSO₄). Filtration and evaporationof solvent afforded the crude product which was purified by flash columnchromatography (99.9:0.1 v/v CHCl₃/MeOH then gradient to 98:2 v/vCHCl₃/MeOH) to afford 26 as a yellow glass (30 mg, 39%): ¹H NMR (400MHz, CDCl₃) 7.86 (d, 2H, J=3.9 Hz, H11), 7.60 (s, 2H, H6), 7.36 (s, 2H,H3), 6.91 (d, 2H, J=0.9 Hz, H_(arom)), 6.86 (s, 2H), 6.82-6.78 (m, 4H,H9 and H_(arom)), 5.97 (s, 2H, OCH₂O), 4.36-4.25 (m, 6H, H11a andOCH₂CH₂CH₂O), 3.93 (s, 6H, OCH₃×2), 3.52 (dd, 2H, J=10.8, 16.6 Hz, H1),3.33 (d, 2H, J=16.6 Hz, H1), 2.46-2.40 (m, 2H, OCH₂CH₂CH₂O).

(c)1,1′-[[(Propane-1,3-diyl)dioxy]bis[(11aS)-11-sulpho-7-methoxy-2-(3,4-methylenedioxyphenyl)-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]] sodiumsalt (30)

A solution of sodium bisulphite (2.2 mg, 0.021 mmol, 2.0 equiv.) in H₂O(0.5 mL) was added to a solution of compound 29 (8.1 mg, 0.0105 mmol) inDCM (0.5 mL). The reaction mixture was allowed to stir for 5 hours. TLC(EtOAc) revealed complete consumption of the starting material and aproduct spot at the base line. The reaction mixture was diluted with DCM(1.5 mL) and H₂O (1.5 mL). The aqueous layer was separated andlyophilized to afford 30 as a lightweight white solid (3.5 mg, 34%): ¹HNMR (400 MHz, DMSO) δ 7.47 (s, 2H, H3), 6.98 (s×2, 2H, H6), 7.06-6.78(m, 6H, H_(arom)), 6.51 (s×2, 2H, H9), 5.27 (s, 2H, NH), 4.32-4.26 (m,2H, H11a), 4.17-4.07 (m, 4H, OCH₂CH₂CH₂O), 3.93 (d, 2H, J=10.4 Hz, H11),3.72 (s, 6H, OCH₃×2), 3.50-3.20 (m, 4H, H1), 2.27-2.20 (m, 2H,OCH₂CH₂CH₂O).

Example 7 (a)1,1′-[[(Propane-1,3-diyl)dioxy]bis[(11S,11aS)-10-(2,2,2-trichloroethoxycarbonyl)-11-(tert-butyldimethylsilyloxy)-7-methoxy-2-(4-fluorobenzene)-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]](31)

A solution of TEA (0.072 mL, 0.52 mmol, 6.0 eq) in H₂O (0.6 mL) and EtOH(3.5 mL) was added to a solution of triflate 13 (125 mg, 0.086 mmol) intoluene (3.5 mL) at room temperature. To this mixture4-fluorobenzeneboronic acid (31.6 0.22 mmol, 2.6 eq) and Pd(PPh₃)₄ (4.0,3.4 μmol, 0.04 eq) were added. The reaction mixture was heated at 100°C. under microwave irradiation for 5 minutes when TLC (80:20 v/vEtOAc/hexane) revealed complete consumption of the starting material.Excess solvent was removed and the residue was dissolved in EtOAc (10mL), washed with H₂O (10 mL), brine (10 mL) and dried (MgSO₄).Filtration and evaporation of solvent afforded the crude product whichwas purified by flash column chromatography (80:20 v/v hexane/EtOAc thengradient to 65:35 v/v hexane/EtOAc) to afford 31 as a yellow glass (82mg, 71%): ¹H NMR (400 MHz, CDCl₃) δ 7.40 (s, 2H, H3), 7.42 (dd, 4H,J=5.2, 8.7 Hz, H_(arom)), 7.28 (s, 2H, H6), 7.06 (dd, 4H, J=8.7 Hz,H_(arom)), 6.70 (s, 2H, H9), 5.92 (d, 2H, J=8.8 Hz, H11), 5.22 (d, 2H,J=12.0 Hz, Troc CH₂), 4.32-4.29 (m, 2H, OCH₂CH₂CH₂O), 4.19-4.10 (m, 4H,Troc CH₂ and OCH₂CH₂CH₂O), 4.00-3.92 (m, 8H, H11a and OCH₃×2), 3.33 (dd,2H, J=10.8, 16.5 Hz, H1), 2.84 (d, 2H, J=16.5 Hz, H1), 2.49-2.44 (m, 2H,OCH₂CH₂CH₂O), 0.92 (s, 18H, TBS CH₃×6), 0.30 and 0.27 (s×2, 12H, TBSCH₃×4).

(b)1,1′-[[(Propane-1,3-diyl)dioxy]bis[(11aS)-7-methoxy-2-(4-fluorobenzene)-1,11a-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]](32)

10% Cd/Pb couple (342 mg, 2.80 mmol, 25 eq) was added to a rapidlystirring mixture of 31 (149 mg, 0.112 mmol), THF (4.5 mL) and 1N NH₄OAc(4.5 mL). The reaction mixture was allowed to stir for 5 hours when TLCshowed complete consumption of the starting material. The solids werefiltered and rinsed with H₂O and MeOH. Excess solvent was removed andthe residue was diluted with H₂O (30 mL) and DCM (30 mL). The aqueouslayer was extracted with DCM (3×30 mL) and the organic extracts werecombined, washed with H₂O (80 mL) and brine (80 mL) and dried (MgSO₄).Filtration and evaporation of solvent afforded the crude product whichwas purified by flash column chromatography (99.9:0.1 v/v CHCl₃/MeOHthen gradient to 98:2 v/v CHCl₃/MeOH) to afford 32 as a yellow glass (60mg, 75%): ¹H NMR (400 MHz, CDCl₃) 7.88 (d, 2H, J=3.9 Hz, H11), δ 7.51(s, 2H, H6), 7.43 (s, 2H, H3), 7.34 (dd, 4H, J=5.3, 8.6 Hz, H_(arom)),7.06 (dd, 4H, J=8.7 Hz, H_(arom)), 6.87 (s, 2H, H9), 4.41-4.25 (m, 6H,H11a and OCH₂CH₂CH₂O), 3.93 (s, 3H, OCH₃×2), 3.55 (dd, 2H, J=10.8, 16.5Hz, H1), 3.37 (d, 2H, J=16.5 Hz, H1), 2.46-2.42 (m, 2H, OCH₂CH₂CH₂O).

(c)1,1′-[[(Propane-1,3-diyl)dioxy]bis[(11aS)-11-sulpho-7-methoxy-2-(4-fluorobenzene)-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]] sodiumsalt (33)

A solution of sodium bisulphite (13.9 mg, 0.134 mmol, 2.0 equiv.) in H₂O(3 mL) was added to a solution of compound 32 (48.0 mg, 0.067 mmol) inDCM (3 mL). The reaction mixture was allowed to stir for 2 hours. TLC(EtOAc) revealed complete consumption of the starting material and aproduct spot at the base line. The reaction mixture was diluted with DCM(8 mL) and H₂O (8 mL). The aqueous layer was separated and lyophilizedto afford 33 as a lightweight white solid (35.5 mg, 58%): ¹H NMR (400MHz, DMSO) δ 7.56 (s, 2H, H3), 7.49 (dd, 4H, J=5.3, 8.6 Hz, H_(arom)),7.17 (dd, 4H, J=8.7 Hz, H_(arom)), 7.07 (s, 2H, H6), 6.53 (s, 2H, H9),5.28 (s, 2H, NH), 4.36-4.30 (m, 2H, H11a), 4.17-4.09 (m, 4H,OCH₂CH₂CH₂O), 3.95 (d, 2H, J=10.4 Hz, H11), 3.73 (s, 6H, OCH₃×2),3.53-3.22 (m, 4H, H1), 2.27-2.19 (m, 2H, OCH₂CH₂CH₂O).

Example 8 (a)1,1′-[[(Pentane-1,5-diyl)dioxy]bis[(2S,4R)-(5-methoxy-2-nitro-1,4-phenylene)carbonyl]]bis[2-(tert-butyldimethylsilyloxymethyl)-4-hydroxyacetylpyrrolidine](36)

A catalytic amount of DMF (2 drops) was added to a stirred solution ofthe nitro-acid 34 (15.0 g, 30.4 mmol) and oxalyl chloride (13.5 mL, 19.3g, 152 mmol) in dry THF (300 mL). The reaction mixture was allowed tostir for 16 hours at room temperature and the solvent was removed byevaporation in vacuo. The resulting residue was re-dissolved in dry THF(225 mL) and the acid chloride solution was added dropwise to a stirredmixture of the amine 35 (20.7 g, 76.0 mmol) and TEA (84 mL, 61.52 g, 608mmol) in THF (150 mL) at 0° C. (ice/acetone) under a nitrogenatmosphere. The reaction mixture was allowed to warm to room temperatureand stirred for a further 16 hours. Excess THF was removed by rotaryevaporation and the resulting residue was partitioned between H₂O (400mL) and EtOAc (400 mL). The layers were allowed to separate and theaqueous layer was extracted with EtOAc (3×130 mL). The combined organiclayers were then washed with saturated NH₄Cl (200 mL), saturated NaHCO₃(200 mL), brine (200 mL) and dried (MgSO₄). Filtration and evaporationof the solvent gave the crude product as a dark coloured oil.Purification by flash chromatography (gradient elution: 60:40 v/vHexane/EtOAc to 40:60 v/v Hexane/EtOAc) isolated the pure amide 36 as alight yellow coloured glass (23.56 g, 77%): ¹H NMR (400 MHz, CDCl₃)(rotamers) δ 7.66 (s, 2H), 6.71 (s, 2H), 5.21-5.17 (m, 2H), 4.57-4.53(m, 2H), 4.21-4.08 (m, 6H), 3.91 (s, 6H), 3.76-3.72 (m, 2H), 3.46 (dd,2H, J=11.8, 4.6 Hz), 3.21 (d, 2H, J=11.9 Hz), 2.44-2.37 (m, 2H),2.24-2.18 (m, 2H), 2.03-1.94 (m, 10H), 1.75-1.67 (m, 2H), 0.91-0.84 (m,18H), 0.11-0.05 (m, 12H); ¹³C NMR (100 MHz, CDCl₃) δ 171.0, 166.4,154.4, 148.5, 137.5, 127.7, 109.2, 108.3, 72.9, 69.3, 62.6, 57.4, 56.5,54.8, 33.0, 28.5, 25.8, 22.5, 21.0, 18.2, −5.4 and −5.5; LC/MS 2.93 min(ES+) m/z (relative intensity) 1006 ([M+H]⁺, 100).

(b)1,1′-[[(Pentane-1,5-diyl)dioxy]bis[(2S,4R)-(5-methoxy-2-amino-1,4-phenylene)carbonyl]]bis[2-(tert-butyldimethylsilyloxymethyl)-4-hydroxyacetylpyrrolidine](37)

A slurry of 10% Pd—C (550 mg) in EtOAc (20 mL) was added to a solutionof the nitro compound 36 (5.5 g, 5.47 mmol) in EtOAc (73 mL). Themixture was subjected to hydrogenation using Parr apparatus at 30 psifor a total of 24 hours. The mixture was degassed and analysed by TLC(EtOAc) where complete consumption of starting material was observed.The catalyst was removed by vacuum filtration through Whatman GF/Ffilter paper and the filtrate evaporated to provide the aniline 37 as agrey coloured foam (5.17 g, 100%): ¹H NMR (400 MHz, CDCl₃) δ 6.71 (s,2H), 6.22 (s, 2H), 5.25-5.23 (m, 2H), 4.58-4.30 (m, 6H), 4.14-4.05 (m,2H), 3.99 (t, J=6.6 Hz, 4H), 3.79-3.73 (m, 8H), 3.69-3.54 (m, 4H),2.40-2.33 (m, 2H), 2.16-2.08 (m, 2H), 2.00 (s, 6H), 1.94-1.87 (m, 4H),1.68-1.60 (m, 2H), 0.89 (s, 18H), 0.05 and 0.04 (s×2, 12H); ¹³C NMR (100MHz, CDCl₃) δ 170.6, 170.0, 151.5, 141.7, 141.2, 113.1, 111.0, 102.0,73.5, 68.5, 62.6, 57.0, 56.2, 32.9, 28.7, 25.6, 22.6, 21.1, 18.1, −5.1and −5.4; LC/MS 2.78 min (ES+) m/z (relative intensity) 946 ([M+H]⁺,52), 672 (30), 399 (20), 274 (65), 166 (20).

(c)1,1′-[[(Pentane-1,5-diyl)dioxy]bis[(2S,4R)-[5-methoxy-1,4-phenylene-2-(2,2,2-trichloroethoxycarbonylamino)]carbonyl]]bis[2-(tert-butyldimethylsilyloxymethyl)-4-hydroxyacetylpyrrolidine](38)

A solution of 2,2,2-trichloroethyl chloroformate (2.63 mL, 4.05 g, 19.1mmol) in dry DCM (45 mL) was added dropwise to a stirred solution ofamine 37 (8.21 g, 8.70 mmol) and anhydrous pyridine (2.81 mL, 2.75 g,34.8 mmol) in dry DCM (120 mL) at −10° C. (liquid N₂/ethanediol). After16 hours stirring at room temperature TLC (50:50 v/v Hexane/EtOAc)revealed complete consumption of starting material. The reaction mixturewas washed with saturated NH₄Cl (2×70 mL), saturated CuSO₄ (70 mL), H₂O(70 mL), brine (70 mL) and dried (MgSO₄). Filtration and evaporation ofthe solvent yielded the Troc-carbamate 38 as a yellow foam (11.25 g,99%): ¹H NMR (400 MHz, CDCl₃) δ 9.48 (br s, 1H), 7.85 (s, 2H), 6.79 (s,2H), 5.28-5.24 (m, 2H), 4.86-4.76 (m, 4H), 4.70-4.53 (m, 2H), 4.12 (t,J=6.4 Hz, 4H), 4.14-4.10 (m, 2H), 3.80 (s, 6H), 3.76 (dd, 2H, J=12.2,3.8 Hz), 3.69-3.62 (m, 4H), 2.44-2.37 (m, 2H), 2.16 (dd, 2H, J=14.1, 8.1Hz), 2.10-1.92 (m, 10H), 1.72-1.65 (m, 2H), 0.90 (s, 18H), 0.06 and 0.04(s×2, 12H); ¹³C NMR (100 MHz, CDCl₃) δ 170.5, 169.3, 152.0, 151.1,144.2, 132.4, 114.4, 111.8, 105.2, 95.3, 74.4, 73.4, 68.7, 62.3, 57.3,57.2, 56.5, 32.6, 28.7, 25.8, 22.6, 21.1, 18.1, −5.4 and −5.5; LC/MS3.23 min (ES+/ES−) m/z (M⁺ not observed), 472 (30), 416 (15), 302 (85),274 (60), 198 (20), 170 (100), 142 (50), 110 (80).

(d)1,1′-[[(Pentane-1,5-diyl)dioxy]bis[(2S,4R)-[5-methoxy-1,4-phenylene-2-(2,2,2-trichloroethoxycarbonylamino)]carbonyl]]bis(2-hydroxymethyl-4-hydroxyacetylpyrrolidine)(39)

Glacial acetic acid (90 mL) was added to a stirred solution of 38 (5.06g, 3.91 mmol) in H₂O (30 mL) and THF (30 mL). The reaction mixture wasallowed to stir at room temperature for 16 h at which point TLC (95:5v/v CHCl₃/MeOH) revealed completion of the reaction. The acidic solutionwas added dropwise to a stirred solution of NaHCO₃ (132 g) in H₂O (1.3L) at 0° C. (ice/acetone). The aqueous layer was extracted with EtOAc(3×200 mL) and the organic layers were combined, washed with H₂O (200mL), brine (200 mL) and dried (MgSO₄). Filtration and evaporation of thesolvent in vacuo afforded the crude product which was purified by flashcolumn chromatography (98:2 v/v CHCl₃/MeOH) to provide the bis-alcohol39 as a white glass (4.04 g, 97%): ¹H NMR (400 MHz, CDCl₃) δ 9.01 (br s,2H), 7.66 (s, 2H), 6.79 (s, 2H), 5.23-5.20 (m, 2H), 4.84 (d, 2H, J=12.0Hz), 4.77 (d, 2H, J=12.0 Hz), 4.63-4.55 (m, 2H), 4.10 (t, J=6.4 Hz, 4H),4.08-4.01 (m, 2H), 3.82 (s, 6H), 3.75 (dd, 2H, J=12.6, 3.7 Hz),3.70-3.58 (m, 4H), 2.26 (dd, 2H, J=14.1, 7.6 Hz), 2.15-2.06 (m, 2H),2.01 (s, 6H), 1.98-1.92 (m, 4H), 1.74-1.65 (m, 2H); ¹³C NMR (100 MHz,CDCl₃) 170.6, 170.4, 152.1, 150.9, 144.8, 131.0, 115.6, 111.3, 105.7,95.3, 74.3, 72.5, 68.7, 64.3, 58.8, 56.6, 56.5, 33.6, 28.5, 22.6, 21.1.

(e) 1,1′-[[(Pentane-1,5-diyl)dioxy]bis[(11S,11aS,2R)-10-(2,2,2-trichloroethoxycarbonyl)-11-hydroxy-7-methoxy-2-hydroxyacetyl-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]](40)

TEMPO (234 mg, 1.50 mmol) and BAIB (5.31 g, 16.5 mmol) were added to astirred solution of bis-alcohol 39 (4.0 g, 3.75 mmol) in DCM (140 mL).The reaction mixture was allowed to stir under a nitrogen atmosphere for24 hours at which point TLC (95:5 v/v CHCl₃/MeOH) revealed conversion ofstarting material to product. The mixture was diluted with DCM (30 mL),washed with saturated aqueous NaHSO₃ (2×30 mL), saturated aqueous NaHCO₃(2×30 mL), brine (30 mL) and dried (MgSO₄). Filtration and evaporationof the solvent afforded the crude product which was purified by flashcolumn chromatography (gradient elution: CHCl₃ to 99.5:0.5 v/vCHCl₃/MeOH) to provide the cyclised product 40 as a light colouredyellow glass (1.99 g, 50%): ¹H NMR (400 MHz, CDCl₃) δ 7.27 (s, 2H), 6.78(s, 2H), 5.68 (d, 2H, J=9.7 Hz), 5.41-5.35 (m, 2H), 5.24 (d, 2H, J=12.0Hz), 4.23 (d, 2H, J=12.0 Hz), 4.14-3.97 (m, 6H), 3.93 (s, 6H), 3.77-3.69(m, 4H), 2.46-2.33 (m, 4H), 2.05 (s, 6H), 2.03-1.87 (m, 4H), 1.70-1.62(m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 170.4, 167.5, 154.4, 150.6, 149.0,127.3, 124.5, 113.7, 110.8, 95.0, 87.5, 75.0, 71.4, 68.8, 58.3, 56.2,51.1, 35.8, 28.5, 22.4, 21.0.

(f)1,1′-[[(Pentane-1,5-diyl)dioxy]bis[(11S,11aS,2R)-10-(2,2,2-trichloroethoxycarbonyl)-11-(tert-butyldimethylsilyloxy)-7-methoxy-2-hydroxyacetyl-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-[1,4]benzodiazepin-5-one]](41)

TBSOTf (1.27 mL, 1.47 g, 5.54 mmol) was added to a stirred solution ofbis-alcohol 40 (1.96 g, 1.85 mmol) and 2,6-lutidine (0.86 mL, 0.79 g,7.39 mmol) in dry DCM (40 mL). The reaction mixture was allowed to stirunder a N₂ atmosphere for 5 hours after which time TLC (EtOAc) revealedconsumption of starting material. Following dilution with DCM (50 mL),the organic mixture was washed with saturated CuSO₄ (30 mL), saturatedNaHCO₃ (30 mL), brine (50 mL) and dried (MgSO₄). Filtration andevaporation of the solvent in vacuo afforded the crude product which waspurified by flash column chromatography (40:60 v/v Hexane/EtOAc) toprovide the product 41 as a white glass (1.31 g, 55%): ¹H NMR (400 MHz,CDCl₃) δ 7.27 (s, 2H), 6.72 (s, 2H), 5.76 (d, 2H, J=9.0 Hz), 5.37 (br s,2H), 5.24 (d, 2H, J=12.1 Hz), 4.17 (d, 2H, J=12.1 Hz), 4.14-3.96 (m,6H), 3.93 (s, 6H), 3.75-3.66 (m, 4H), 2.40-2.33 (m, 2H), 2.28-2.18 (m,2H), 2.04 (s, 6H), 1.98-1.90 (m, 4H), 1.69-1.62 (m, 2H), 0.86 (s, 18H),0.23 and 0.22 (s×2, 12H); ¹³C NMR (100 MHz, CDCl₃) δ 170.4, 168.0,153.6, 150.5, 149.1, 127.9, 125.3, 113.9, 110.7, 95.2, 88.2, 74.7, 71.7,68.7, 60.5, 56.2, 51.2, 36.3, 28.7, 25.6, 22.7, 21.1, 17.8, −4.2 and−5.2.

(g)1,1′-[[(Pentane-1,5-diyl)dioxy]bis[(11S,11aS,2R)-10-(2,2,2-trichloroethoxycarbonyl)-11-(tert-butyldimethylsilyloxy)-7-methoxy-2-hydroxy-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]](42)

A solution of K₂CO₃ (685 mg, 4.96 mmol) in H₂O (15 mL) was added to astirred solution of acetate compound 41 (1.28 g, 0.99 mmol) in MeOH (15mL). The initial colourless solution eventually turned yellow and theformation of a white precipitate was observed. The reaction mixture washeated at reflux for 5 hours at which point TLC (EtOAc) revealedcomplete conversion of starting material to product. Excess solvent wasremoved by rotary evaporation and the mixture was carefully neutralizedwith 1N HCl to pH 7. The resulting mixture was extracted with DCM (5×20mL) and the combined organic layers were then washed with brine (50 mL)and dried (MgSO₄). Filtration followed by removal of the solvent byevaporation in vacuo afforded the product 42 as a white solid (982 mg,82%): ¹H NMR (400 MHz, CDCl₃) δ 7.19 (s, 2H), 6.72 (s, 2H), 5.75 (d, 2H,J=9.0 Hz), 5.22 (d, 2H, J=12.0 Hz), 4.58-4.56 (m, 2H), 4.18 (d, 2H,J=12.0 Hz), 4.11-3.95 (m, 6H), 3.85 (s, 6H), 3.75-3.69 (m, 2H), 3.59(dd, 2H, J=12.7, 4.2 Hz), 2.55 (br s, 2H), 2.38-2.25 (m, 2H), 2.14-2.03(m, 2H), 1.97-1.85 (m, 4H), 1.74-1.62 (m, 2H), 0.86 (s, 18H), 0.22 and0.21 (s×2, 12H); ¹³C NMR (100 MHz, CDCl₃) δ 168.5, 153.6, 150.6, 149.1,127.9, 125.3, 114.0, 110.8, 95.3, 88.3, 74.7, 69.5, 68.8, 60.8, 56.0,54.0, 38.8, 28.8, 25.6, 22.7, 17.8, −4.2 and −5.2.

(h) 1,1′-[[(Pentane-1,5-diyl)dioxy]bis[(11S,11aS)-10-(2,2,2-trichloroethoxycarbonyl)-11-(tert-butyldimethylsilyloxy)-7-methoxy-2-oxo-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]](43)

A solution of anhydrous DMSO (0.34 mL, 0.37 g, 4.78 mmol) in dry DCM (4mL) was added dropwise over a period of 5 minutes to a stirred solutionof oxalyl chloride (1.20 mL of a 2M solution in DCM, 2.39 mmol) under anitrogen atmosphere at −60° C. (liq N₂/CHCl₃). After stirring at −55° C.for 15 minutes, a slurry of the substrate 42 (962 mg, 0.80 mmol) in dryDCM (8 mL) was added dropwise over a period of 10 minutes to thereaction mixture. After stirring for a further 1 h at −55° C., asolution of TEA (1.56 mL, 1.13 g; 11.2 mmol) in dry DCM (4 mL) was addeddropwise over a period of 5 minutes to the reaction mixture. Thereaction mixture was allowed to warm to 0° C. and then diluted with DCM(50 mL). The organic solution was washed with cold 1N HCl (20 mL), H₂O(20 mL), brine (30 mL) and dried (MgSO₄). Filtration and evaporation ofthe solvent in vacuo afforded the crude product which was purified byflash column chromatography (50:50 v/v Hexane/EtOAc) to affordbis-ketone 43 as a foam (550 mg, 57%): ¹H NMR (400 MHz, CDCl₃) δ 7.25(s, 2H), 6.76 (s, 2H), 5.82 (d, 2H, J=9.3 Hz), 5.22 (d, 2H, J=12.0 Hz),4.32 (d, 2H, J=20.4 Hz), 4.22 (d, 2H, J=12.0 Hz), 4.17-4.08 (m, 4H),4.03-3.89 (m, 10H), 2.96 (dd, 2H, J=19.6, 10.2 Hz), 2.56 (dd, 2H,J=19.6, 2.8 Hz), 1.99-1.92 (m, 4H), 1.72-1.64 (m, 2H), 0.86 (s, 18H),0.24 and 0.23 (s×2, 12H); ¹³C NMR (100 MHz, CDCl₃) δ 207.7, 168.0,153.8, 151.0, 149.5, 127.9, 124.4, 114.1, 110.8, 95.2, 87.5, 74.8, 68.8,58.9, 56.2, 52.9, 40.4, 28.7, 25.6, 22.8 17.8, −4.12 and −5.31.

(i) 1,1′-[[(Pentane-1,5-diyl)dioxy]bis[(11S,11aS)-10-(2,2,2-trichloroethoxycarbonyl)-11-(tert-butyldimethylsilyloxy)-7-methoxy-2-[[(trifluoromethyl)sulfonyl]oxy]-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]](44)

Anhydrous triflic anhydride taken from a freshly opened ampoule (1.61mL, 2.71 g, 9.60 mmol) was added rapidly in one portion to a vigorouslystirred solution of ketone 43 (525 mg, 0.44 mmol) and anhydrous pyridine(0.78 mL, 759 mg, 9.60 mmol) in dry DCM (25 mL) at 0-5° C. (ice) under anitrogen atmosphere. The reaction mixture was immediately allowed towarm to room temperature and eventually turned a dark red colour. Thereaction mixture was allowed to stir for a total of 28 hours at whichpoint TLC (80:20 v/v EtOAc/hexane) revealed the complete consumption ofstarting material. The mixture was poured into cold saturated NaHCO₃ (50mL) and extracted with DCM (3×20 mL). The combined organic layers werethen washed with saturated CuSO₄ (30 mL), brine (30 mL) and dried(MgSO₄). Filtration and evaporation of the solvent in vacuo afforded thecrude product which was purified by flash column chromatography (80:20v/v Hexane/EtOAc) to afford triflate 44 as a yellow foam (249 mg, 39%):¹H NMR (400 MHz, CDCl₃) δ 7.23 (s, 2H), 7.17 (s, 2H), 6.73 (s, 2H), 5.93(d, 2H, J=8.9 Hz), 5.22 (d, 2H, J=12.1 Hz), 4.21 (d, 2H, J=12.0 Hz),4.15-4.07 (m, 2H), 4.02-3.86 (m, 8H), 3.33 (ddd, 2H, J=16.6, 10.7, 2.3Hz), 2.82 (dd, 2H, J=16.7, 2.6 Hz), 1.98-1.91 (m, 4H), 1.71-1.63 (m,2H), 0.88 (s, 18H), 0.28 and 0.25 (s×2, 12H); ¹³C NMR (100 MHz, CDCl₃) δ164.9, 153.6, 151.3, 149.6, 136.0, 127.8, 123.7, 121.0, 118.6 (q,J=321.5 Hz), 114.2, 111.0, 95.1, 86.4, 74.9, 68.8, 60.6, 56.2, 34.4,28.7, 25.6, 22.8, 17.8, −4.2 and −5.4.

(j)1,1′-[[(Pentane-1,5-diyl)dioxy]bis[(11S,11aS)-10-(2,2,2-trichloroethoxycarbonyl)-11-(tert-butyldimethylsilyloxy)-7-methoxy-2-(p-methoxyphenyl)-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]](45)

Pd(PPh₃)₄ (7.43 mg, 6.43 μmol) was added to a solution of the triflate44 (236 mg, 0.16 mmol), 4-methoxyphenylboronic acid (63 mg, 0.42 mmol),TEA (0.13 mL, 98 mg, 0.97 mmol) in H₂O (0.8 mL), EtOH (5 mL) and toluene(5 mL) at room temperature. The reaction mixture was heated in themicrowave at 100° C. for 30 minutes after which time TLC (80:20 v/vEtOAc/hexane) revealed the complete consumption of the startingmaterial. Excess solvent was removed by evaporation in vacuo and theresulting residue was dissolved in EtOAc (50 mL), washed with H₂O (20mL), brine (20 mL) and dried (MgSO₄). Filtration and evaporation ofsolvent afforded the crude product which was purified by flash columnchromatography (gradient elution: 80:20 v/v Hexane/EtOAc to 50:50 v/vHexane/EtOAc) to afford C2-aryl product 45 as a yellow glass (117 mg,52%): ¹H NMR (400 MHz, CDCl₃) δ 7.35 (s, 2H), 7.31 (s, 2H), 7.31-7.27(m, 6H), 6.90 (d, 4H, J=8.8 Hz), 6.76 (s, 2H), 5.92 (d, 2H, J=8.8 Hz),5.23 (d, 2H, J=12.0 Hz), 4.20 (d, 2H, J=12.1 Hz), 4.14-4.05 (m, 4H),4.04-3.95 (m, 2H), 3.93 (s, 6H), 3.83 (s, 6H), 3.34 (ddd, 2H, J=16.3,10.4, 2.0 Hz), 2.86-2.75 (m, 2H), 2.02-1.94 (m, 4H), 1.72-1.66 (m, 2H),0.93 and 0.86 (s×2, 18H), 0.28 and 0.25 (s×2, 12H); ¹³C NMR (100 MHz,CDCl₃) δ 163.7, 159.1, 153.7, 150.8, 149.3, 127.8, 126.3, 126.1, 125.4,122.3, 122.2, 114.3, 114.2, 110.9, 95.3, 87.3, 74.8, 68.8, 61.5, 56.2,55.4, 35.3, 28.8, 25.7, 22.8, 17.9, −4.0 and −5.1.

(k)1,1′-[[(Pentane-1,5-diyl)dioxy]bis[(11aS)-7-methoxy-2-(p-methoxyphenyl)-1,11a-dihydro-5H-pyrrolo[2,1-[1,4]benzodiazepine-5-one]](46)

10% Cd/Pb couple (192 mg, 1.55 mmol) was added to a rapidly stirringmixture of 45 (107 mg, 77.3 μmol), THF (2.5 mL) and 1N NH₄OAc (2.5 mL).The reaction mixture was allowed to stir for 3 hours at which point TLC(95:5 v/v CHCl₃/MeOH) showed the formation of desired PBD accompanied byside-products. The solids were collected by filtration and rinsed withH₂O and DCM. The aqueous layer was extracted with DCM (3×10 mL) and theorganic extracts were combined, washed with brine (50 mL) and dried(MgSO₄). Filtration and evaporation of solvent afforded the crudeproduct which was purified by flash column chromatography (gradientelution: CHCl₃ to 99:1 v/v CHCl₃/MeOH) to afford the imine 46 as ayellow glass (32.5 mg, 55%): ¹H NMR (400 MHz, CDCl₃) δ 7.89 (d, 2H,J=3.9 Hz), 7.53 (s, 2H), 7.39 (s, 2H), 7.34 (d, 4H, J=8.8 Hz), 6.90 (d,4H, J=8.8 Hz), 6.82 (s, 2H), 4.43-4.38 (m, 2H), 4.18-4.05 (m, 4H), 3.95(s, 6H), 3.83 (s, 6H), 3.58 (ddd, 2H, J=16.2, 11.5, 1.9 Hz), 3.38 (ddd,2H, J=16.3, 5.1, 1.6 Hz), 2.01-1.94 (m, 4H), 1.73-1.66 (m, 2H); ¹³C NMR(100 MHz, CDCl₃) δ 162.6, 161.3, 159.2, 151.3, 148.1, 140.3, 126.2,126.0 (×2), 123.2, 122.0, 119.1, 114.3, 111.9, 110.9, 68.8, 56.2, 55.4,53.9, 35.6, 28.7, 22.6.

(j)1,1′-[[(Pentane-1,5-diyl)dioxy]bis[(11aS)-11-sulpho-7-methoxy-2-(p-methoxyphenyl)-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one]]sodium salt (47)

A solution of sodium bisulphite (4.90 mg, 47.2 μmol) in water (3 mL) wasadded to a stirred solution of 46 (18.11 mg, 23.6 μmol) indichloromethane (1.5 mL). The reaction mixture was allowed to stirvigorously for 24 hours, after which time the organic and aqueous layerswere separated. TLC analysis (95:5 v/v CHCl₃/MeOH) of the aqueous phaserevealed absence of 46 (R_(f)˜0.3) and the presence of baseline materialwith strong UV-absorption. The aqueous layer was lyophilised to providethe bisulphite adduct 47 as a lightweight solid (10 mg, 43%): ¹H NMR(400 MHz, DMSO-d₆) δ 7.41 (s, 2H), 7.37 (d, 4H, J=8.7 Hz), 7.04 (s, 2H),6.91 (d, 4H, J=8.9 Hz), 6.47 (s, 2H), 5.24 (s, 2H), 4.31-4.21 (m, 2H),4.06-3.86 (m, 6H), 3.76 (s, 6H), 3.71 (s, 6H), 3.51-3.21 (m, 4H),1.90-1.70 (m, 4H), 1.62-1.51 (m, 2H).

Example 9 Determination of In Vitro Cytotoxicity

K562 Cell Line (MTT Assay)

1 Hour Exposure

K562 human chronic myeloid leukaemia cells were maintained in RPM1 1640medium supplemented with 10% fetal calf serum and 2 mM glutamine at 37°C. in a humidified atmosphere containing 5% CO₂ and were incubated witha specified dose of drug for 1 hour at 37° C. in the dark. Theincubation was terminated by centrifugation (5 min, 300 g) and the cellswere washed once with drug-free medium. Following the appropriate drugtreatment, the cells were transferred to 96-well microtiter plates (10⁴cells per well, 8 wells per sample). Plates were then kept in the darkat 37° C. in a humidified atmosphere containing 5% CO₂. The assay isbased on the ability of viable cells to reduce a yellow solubletetrazolium salt,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT,Aldrich-Sigma), to an insoluble purple formazan precipitate. Followingincubation of the plates for 4 days (to allow control cells to increasein number by approximately 10 fold), 20 μL of MTT solution (5 mg/mL inphosphate-buffered saline) was added to each well and the plates furtherincubated for 5 h. The plates were then centrifuged for 5 min at 300 gand the bulk of the medium pipetted from the cell pellet leaving 10-20μL per well. DMSO (200 μL) was added to each well and the samplesagitated to ensure complete mixing. The optical density was then read ata wavelength of 550 nm on a Titertek Multiscan ELISA plate reader, and adose-response curve was constructed. For each curve, an IC₅₀ value wasread as the dose required to reduce the final optical density to 50% ofthe control value.

96 Hour Exposure

In a modification of the above method, 5×10⁴ K562 cells (as above) weremaintained in RPMI 1640 medium (Sigma) supplemented with 10% foetal calfserum (Sigma) and 20 mM L-glutamine (Sigma) and 190 μL of this solutionincubated with specific doses of the drug (10 μL) for 96 hours at 37° C.in 5% CO₂. 250 μg/ml (final) MTT was added to each well and the platesincubated for a further 4 hours. Absorbance was read at 450 nm using anEnvision plate reader (Applied Biosystems). Data was then analysed byGraphpad PRISM, and an IC₅₀ obtained (defined as the compoundconcentration needed to reduce the cell population by half).

Results

Compound IC₅₀ (μM) ZC-207 0.0053^(a) ZC-423 0.0015^(b) ^(a)1 hourincubation ^(b)96 hour incubationK562 Cell Line (Alamar Blue Assay)

The compounds to be tested were dissolved in 100% Biotech grade DMSO(Sigma) and subsequently diluted to stock concentrations of either 2 μMor 200 nM in 2% DMSO. 100 μl of the stock concentrations were seriallydiluted 1 in 3 in 2% DMSO in intermediate polystyrene 96 well cellculture plates (Nunc). 2% DMSO was placed in the outer columns for useas blanks, and in the 2^(nd) or 11^(th) column (depending on whether thetop or bottom of an assay plate was to be used) for use as a control.The entire row of the intermediate plate was then transferred intriplicate to either rows B to D or E to G of a fluorescence compatiblepolystyrene 96 well cell culture plate (Greiner BioOne); 10 μl to eachwell. A cell solution containing 5×10⁴ cells/ml was made up in phenolred free RPMI 1640 (Sigma) supplemented with 10% foetal calf serum(Sigma) and 20 mM L-glutamine (Sigma). 190 μl cell solution was added toeach well of the assay plate, from columns 2 to 11. 190 μl culture mediawas added to columns 1 and 12. Plates were incubated for 96 hours at 37°C. in 5% CO₂. 1 μM (final) rezasurin (converted to fluorescent resofurinby viable cells) was added to each well and the plates incubated for afurther 4 hours. Fluorescence was read at 530-570 nm excitation, 580-620nm emission using an Envision plate reader (Applied Biosystems). Datawas then analysed by Graphpad PRISM, and an IC₅₀ obtained (defined asthe compound concentration needed to reduce the cell population byhalf).

Results

Compound IC₅₀ (nM) 19 <1 22 <1 26 <1 27 <1 29 <1 30 <1 32 <1 33 <1LOXIMVI and OVCAR-5 Cell Lines

A cell solution containing 5×10⁴ cells/ml of either LOXIMVI humanmelanoma cells or OVCAR-5 human ovarian tumour cells was made up inphenol red free RPMI 1640 (Sigma) supplemented with 10% foetal calfserum (Sigma) and 20 mM L-glutamine (Sigma). 190 μl cell solution wasadded to each well of a fluorescence compatible polystyrene 96 well cellculture plate (Greiner BioOne), from columns 2 to 11. 190 μl culturemedia was added to columns 1 and 12. Plates were incubated overnight at37° C. in 5% CO₂. Compounds were dissolved in 100% Biotech grade DMSO(Sigma) and subsequently diluted to stock concentrations in 2% DMSO. 100μl of the stock concentrations were serially diluted 1 in 3 in 2% DMSOin intermediate polystyrene 96 well cell culture plates (Nunc). 2% DMSOwas placed in the outer columns for use as blanks, and in the 2^(nd) or11^(th) column (depending on whether the top or bottom of an assay platewas to be used) for use as a control. The entire row of the intermediateplate was then transferred in triplicate, 10 μl per well, to either rowsB to D or E to G of the cell plate. Plates were incubated for 96 hoursat 37° C. in 5% CO₂ 1 μM (final) rezasurin (converted to fluorescentresofurin by viable cells) was added to each well and the platesincubated for a further 4 hours. Fluorescence was read at 530-570 nmexcitation, 580-620 nm emission using an Envision plate reader (AppliedBiosystems). Data was then analysed by Graphpad PRISM, and an IC₅₀obtained (defined as the compound concentration needed to reduce thecell population by half).

Results

Cell line ZC-423 IC₅₀ (nM) LOXIMVI 4.83 OVCAR-5 5.2

Example 10 Anti Tumour Activity

The following experiments was carried out under a project licenceapproved by the Home

Office, London, UK, and UK CCCR guidelines (Workman, P., et al., BritishJournal of Cancer, 77, 1-10 (1998)) were followed throughout.

LOX IMVI (human melanomas) were grown subcutaneously in nude mice (B&KUniversal, Hull, UK). Tumours were transplanted as single tumourfragments in the flank by trocar. Groups of 8 tumour-bearing mice weretreated with ZC423 at the previously established single intravenous (iv)maximum tolerated dose (MTD) of 3 mgkg⁻¹ using 5% DMA/saline as thevehicle. Control mice (n=8) were treated with vehicle alone.

Treatment commenced when tumours could be reliably measured by calipers(mean dimensions 4×4 mm) and therapeutic effects were assessed by dailycaliper measurements of the tumour and mouse weights. Tumour volumeswere determined by the formula a²×b/2 where a is the smaller and b isthe larger diameter of the tumour. The results are shown in FIG. 1 as agraphs of relative tumour volume against time (▪ ZC423; ♦Control—solvent alone). The activity of ZC423 was shown to bestatistically significant when assessed by Mann-Whitney analysis (asdescribed, for example, in Essential Statistics 2^(nd) Edition (1989),D. G. Rees, ISBN 0 412 32030 4, Chapman and Hall, London).

FIG. 2 shows the results of the same test (● ZC423; ♦ Control—solventalone) but extended over a longer period, and with a larger scale forthe Mean RTV. The control group was ceased at 16 days, at which pointthere were no tumour free mice, compared to the treated group where allanimals were tumour free at 68 days.

A study was carried out using OVCAR-5 (human ovarian tumours) under thesame conditions as described above. The results are shown in FIG. 3 (●ZC423; ♦ Control—solvent alone). In this study the control group wasceased at 32 days.

Example 11 Cross-Linking and Solubility

DNA Cross-Linking

Closed-circular puc18 DNA was linearized with HindIII, thendephosphorylated, and finally 5′ end labeled with [y32P]-ATP usingpolynucleotide kinase. Reactions containing 10 ng of DNA and drug werecarried out in aqueous 1×TEOA (25 mM triethanolamine, 1 mM EDTA, pH 7.2)buffer at a final volume of 50 μL at 37° C. Reactions were terminated byaddition of an equal volume of stop solution (0.6 M NaOAc, 20 mM EDTA,100 μg/mL tRNA) followed by precipitation with ethanol. Followingcentrifugation of the samples, the supernatants were discarded and thepellets were dried by lyophilization. Samples were resuspended in 10 μLof alkaline denaturing buffer (4 mg bromophenol blue, 600 mg sucrose and40 mg NaOH) and vortexed for three minutes at room temperature. Thenon-denatured controls were re-suspended in 10 μL of standard sucroseloading dye (2.5 mg bromophenol blue, 2.5 mg xylene cyanol blue and 4 gsucrose). Both samples and controls were loaded directly onto an agarosegel.

Electrophoresis was performed on a 0.8% submerged horizontal agarosegel, 20 cm in length for 16 hours at 38-40 V in 1×TAE running buffer(2.42 g Tris Base, 0.372 g EDTA, 0.571 ml glacial acetic acid). Gelswere dried under vacuum for 80 minutes at 80° C. on a Savant SG210DSpeedGel gel dryer onto one layer of Whatman 3MM with a layer of DE81filter paper underneath. An autoradiograph was obtained, after overnightexposure onto FujiRX x-ray film. The film bands were quantitated using aBioRad GS-670 imaging laser densitometer. The percentage ofcross-linking was calculated by measuring the total DNA in each lane(the sum of the densities for the double-stranded and single-strandedbands) relative to the amount of density of double-stranded band alone.A dose response curve was derived by plotting drug concentration againstthe determined percentage level of cross-linked DNA, from which wasderived the amount required to cross-link 50% of the DNA (XL₅₀).

Solubility

Solubility was determined by dissolving the test compound in the minimumamount of water to produce a saturated solution.

Results

DNA Cross-linking

Compound IC₅₀ (μM) 18 0.2 19 2 21 0.5 22 0.3Solubility

Compound Amount of compound (g) in 1 L water ZC-207 Insoluble ZC-423 11 27 9 30 4 33 3

The invention claimed is:
 1. A compound of formula:


2. A pharmaceutical composition comprising a compound according to claim1, and a pharmaceutical excipient.
 3. A method of treating aproliferative disease comprising administering a therapeuticallyeffective amount of a compound according to claim 1, wherein theproliferative disease is selected from the group consisting of leukemia,melanoma, and ovarian cancer.